text:
Tumor necrosis factor (TNF) is associated with the pathophysiology of various neurological disorders, including multiple sclerosis. It exists as a transmembrane form tmTNF, signaling via TNF receptor 2 (TNFR2) and TNFR1, and a soluble form, solTNF, signaling via TNFR1. Multiple sclerosis is associated with the detrimental effects of solTNF acting through TNFR1, while tmTNF promotes repair and remyelination. Here we demonstrate that oligodendroglial TNFR2 is a key mediator of tmTNF-dependent protection in experimental autoimmune encephalomyelitis (EAE). CNP-cre:TNFR2fl/fl mice with TNFR2 ablation in oligodendrocytes show exacerbation of the disease with increased axon and myelin pathology, reduced remyelination, and increased loss of oligodendrocyte precursor cells and mature oligodendrocytes. The clinical course of EAE is not improved by the solTNF inhibitor XPro1595 in CNP-cre:TNFR2fl/fl mice, indicating that for tmTNF to promote recovery TNFR2 in oligodendrocytes is required. We show that TNFR2 drives differentiation of oligodendrocyte precursor cells, but not proliferation or survival. TNFR2 ablation leads to dysregulated expression of microRNAs, among which are regulators of oligodendrocyte differentiation and inflammation, including miR-7a. Our data provide the first direct in vivo evidence that TNFR2 in oligodendrocytes is important for oligodendrocyte differentiation, thereby sustaining tmTNF-dependent repair in neuroimmune disease. Our studies identify TNFR2 in the CNS as a molecular target for the development of remyelinating agents, addressing the most pressing need in multiple sclerosis therapy nowadays.
SIGNIFICANCE STATEMENT Our study, using novel TNF receptor 2 (TNFR2) conditional KO mice with selective TNFR2 ablation in oligodendrocytes, provides the first direct evidence that TNFR2 is an important signal for oligodendrocyte differentiation. Following activation by transmembrane TNF, TNFR2 initiates pathways that drive oligodendrocytes into a reparative mode contributing to remyelination following disease. This identifies TNFR2 as a new molecular target for the development of therapeutic agents in multiple sclerosis.
Multiple sclerosis (MS) is the most common neurological disease in young adults. Despite the general consensus on the autoimmune component of MS, its etiology remains unknown. Current treatments can slow disease progression, reduce relapses, and improve quality of life, but are not effective in halting or reverting myelin and axon damage, which are causes of permanent disability. To address the need for long-term reparative therapies, new pharmacological targets must be identified by elucidating the mechanisms of the underlying neurodegenerative process.
Tumor necrosis factor (TNF) is an immunomodulatory cytokine regulating physiological and pathological processes. TNF exists in two forms, transmembrane TNF (tmTNF) and soluble TNF (solTNF). solTNF derives from the cleavage of tmTNF by the TNF-α converting enzyme (Kriegler et al., 1988). The cellular functions of TNF are mediated by TNF receptor 1 (TNFR1) and TNFR2, which differ in expression, ligand affinity, structure, and downstream signaling pathways. solTNF and tmTNF can bind to both receptors but with different affinities. As a result, solTNF signals only through TNFR1 mediating primarily inflammation and apoptosis (Holtmann and Neurath, 2004), while tmTNF signals through both TNFR1 and TNFR2, promoting cell survival, resolution of inflammation, immunity and myelination (Grell et al., 1995; Probert, 2015).
TNF has been linked to MS pathophysiology (McCoy and Tansey, 2008). MS patients have high concentrations of solTNF in serum and CSF (Maimone et al., 1991; Sharief and Hentges, 1991), and marked TNF immunoreactivity within active lesions (Hofman et al., 1989; Selmaj et al., 1991). In experimental models of MS, TNF overexpression causes demyelination (Probert et al., 1995; Akassoglou et al., 1998; Dal Canto et al., 1999), while blockade improves the outcome (Ruddle et al., 1990; Baker et al., 1994). Nevertheless, the only MS clinical trial with a nonselective TNF inhibitor, Lenercept, had to be terminated due to occurrences of demyelination (Lenercept Multiple Sclerosis Study Group and University of British Columbia MS/MRI Analysis Group, 1999). Subsequent studies in knock-out (KO) mice uncovered that the two forms of TNF and their receptors have opposing biologic effects. Ablation of TNF or TNFR1/TNFR2 combined did not protect from experimental autoimmune encephalomyelitis (EAE), rather it caused exacerbation of chronic disease (Körner et al., 1997; Liu et al., 1998; Eugster et al., 1999; Kassiotis et al., 1999; Suvannavejh et al., 2000). TNFR1 ablation resulted in less severe EAE and better remyelination, while TNFR2 ablation caused worse EAE and prevented remyelination (Liu et al., 1998; Eugster et al., 1999; Suvannavejh et al., 2000; Arnett et al., 2001). Furthermore, mice only expressing tmTNF showed suppression of EAE (Alexopoulou et al., 2006). Based on these studies, the understanding of TNF involvement in MS has evolved, and a dichotomy has emerged between solTNF and tmTNF: MS and EAE are associated with the detrimental effects of solTNF via TNFR1, while tmTNF is important for repair and remyelination via TNFR2. In our own studies, we demonstrated that selective blockade of solTNF with XPro1595 improved EAE outcome, while the inhibition of both TNF forms with etanercept exacerbated the disease (Brambilla et al., 2011). The therapeutic effect of XPro1595 was associated with axon preservation, improved myelin compaction, and increased remyelination, suggesting that tmTNF might have protective functions by acting directly on cells of the oligodendrocyte lineage, which we showed express high levels of the tmTNF-responsive receptor TNFR2. To address this hypothesis, we generated oligodendrocyte-specific TNFR2 conditional KO mice (CNP-cre:TNFR2fl/fl mice). CNP-cre:TNFR2fl/fl mice exhibit more severe EAE, which is not improved by XPro1595 treatment, indicating that oligodendroglial TNFR2 is required for tmTNF beneficial functions. We show that, without TNFR2, oligodendrocyte differentiation is impaired and microRNAs (miRNAs) known to be crucial signals in regulating this process (miR-219, miR-138, miR-338, miR-7a) are dysregulated. Our study demonstrates that TNF signaling via TNFR2 has important repair functions in demyelinating disease by directly promoting oligodendrocyte differentiation. This has significant implications, particularly for the chronic progressive form of MS, for which no treatments are available. Enhancing TNFR2 signaling in the CNS may provide a viable therapeutic option to boost remyelination and achieve neuroprotection, halting, or even reverting, the progression of the disease.
Materials and Methods
Adult, age-matched (2–4 months of age) female and male mice were used in this study: males for all behavioral assessments in naive conditions, females for all EAE experiments, as they develop a more reproducible disease clinical course. All mouse lines were on a C57BL/6 background, since this genetic background develops a chronic nonrelapsing form of disease when used in myelin oligodendrocyte glycoprotein 35–55 peptide (MOG35–55)-induced EAE (Rangachari and Kuchroo, 2013). TNFR2fl/fl mice were obtained by breeding C57BL/6NTac-Tnfrsf1btm1a(EUCOMM)Wtsi/Ics mice (European Mouse Mutant Archive, EUCOMM Consortium) with an FLP deleter strain (catalog #009086, The Jackson Laboratory) to remove the FRT (FLP recombinase target)-flanked neomycin cassette. CNP-cre+/− mice (Lappe-Siefke et al., 2003) were a gift from Dr. Klaus Nave (Max Planck Institute of Experimental Medicine, Gottingen, Germany). TNFR2fl/fl and CNP-cre+/− mice were bred to generate CNP-cre:TNFR2fl/fl conditional KOs. TNFR2fl and TNFR2WT alleles were genotyped with the following primers: forward, 5′ TTGGGTCTAGAGGTGGCGCAGC 3′; and reverse, 5′ GGCCAGGAAGTGGGTTACTTTAGGGC 3′. TNFR2−/− (catalog #002620), WT C57BL/6, and Rosa26-stop-EYFP mice (catalog #006148) were obtained from The Jackson Laboratory. Colonies were housed in the Animal Core Facility of The Miami Project to Cure Paralysis, in a virus/antigen-free vivarium with a 12 h light/dark cycle, controlled temperature and humidity, and were provided with water and food ad libitum. Mice were group caged (maximum five per cage) throughout the duration of the experimentations. Experiments were performed according to protocols and guidelines approved by the Institutional Animal Care and Use Committee of the University of Miami, and all efforts were made to minimize pain and distress.
Open field test.
The open field test was performed on adult male mice in an odor-free, nontransparent square arena, as previously described (Madsen et al., 2015). The arena was divided into three zones (wall, intermediate, and center) and mouse behavior was recorded over a 5 min period using a high-resolution video camera. The total number of lines crossed; the time spent in each zone; and stereotypical behaviors, such as grooming and rearing, were analyzed and expressed as the number of events. Mice that did not enter all three zones or cross a minimum of 50 lines during the 5 min trial were excluded.
Motor coordination and balance were tested on the accelerating rotarod cylinder (Rotamex 4/8, Columbus Instruments) in adult male mice, as previously described (Madsen et al., 2015). The test consisted of a 5 d pretraining (days 1–5) followed by the actual test (day 7). The cylinder rotated at increasing speed and constant acceleration (from 10 to 60 rpm over a 10 min period). The total time spent on the rod before falling was recorded, and nonwalking behaviors, such as passive clinging to the rod, were manually corrected for. Each trial consisted of an average of 4 sessions. After each session, the mice were transferred back to their cage and allowed to rest for 20 min to avoid exhaustion and minimize stress. Mice that could not maintain their balance on the rod for a minimum of 60 s during pretraining were excluded.
Induction of EAE
Active EAE was induced in 2-month-old female mice with MOG35–55 (BioSynthesis), as previously described (Brambilla et al., 2014). In select experiments, mice were treated with XPro1595 (10 mg/kg, s.c., every 3 d; Xencor) or vehicle (0.9% saline). Behavioral assessments and drug administration were conducted by a blinded investigator, as previously described (Brambilla et al., 2014).
Mice were perfused with 0.1 m PBS followed by 4% paraformaldehyde (PFA) in PBS. Spinal cord and brain were dissected out and postfixed for 2 h in 4% PFA. Tissues were cryoprotected in PBS plus 20% sucrose and cut with a cryostat into 30-μm-thick serial sections. After blocking for 1 h with 5% normal goat serum in PBS plus 0.4% Triton-X, sections were incubated overnight at 4°C with primary antibodies against APC (CC1 clone; 1:500; catalog #OP80, Millipore), NG2 (1:200; catalog #AB5320, Millipore), PDGF receptor α (PDGFRα; 1:200; catalog #558774, BD Bioscience), Olig2 (1:500; #AB9610, Millipore), GFP (1:1000; catalog #13970, Abcam), TNFR2 (1:50; catalog #SC-8041 and #SC-7862, Santa Cruz Biotechnology). Immunoreactivity was visualized with secondary species-specific fluorescent antibodies (1:750; Alexa Fluor-488, Alexa Fluor-594, and Alexa Fluor-647, Invitrogen). Sections were coverslipped with Vectashield mounting medium with DAPI (Vector Laboratories), and imaged with an Olympus FluoView 1000 confocal microscope or with a Zeiss Axiovert A1 fluorescence microscope.
Five 30-μm-thick serial sections taken at 300 μm intervals were analyzed at a 63× magnification with a Zeiss Axiovert A1 fluorescence microscope by an investigator blinded to the genotype. The number of positive cells per cubic millimeter of tissue was estimated with Stereo Investigator software (MBF Bioscience) using the Optical Fractionator for unbiased counting and systematic random sampling.
Toluidine blue analysis and electron microscopy
PFA-fixed 1 mm segments of the thoracic spinal cord were postfixed overnight in 2% glutaraldehyde plus 100 mm sucrose in 0.15 m phosphate buffer, before incubation with 2% OsO4 for 1 h. Following dehydration in graded ethanol solutions, tissues were embedded in epoxy resin (Embed, Electron Microscopy Sciences). Semithin sections (1 μm thick) were obtained with a Leica Ultracut E microtome and stained with 1% toluidine blue solution. Samples were then examined by light microscopy. The number of toluidine blue-stained myelinated or degenerated axons was estimated using Stereo Investigator software (MBF Bioscience). From the same specimens, ultrathin sections (60–90 nm thick) were cut for electron microscopy (EM) and imaged with a Philips CM-10 transmission electron microscope.
Assessment of g-ratio and remyelination
EM micrographs of the spinal cord white matter were taken at a 5.2 thousand magnification (Brambilla et al., 2011). A grid was placed over the section, and pictures were taken randomly from each quadrant. One picture per quadrant was evaluated for a minimum of 25 images/mouse. Fiber and axon diameter of each axon were measured with ImageJ. Axons identified as “remyelinating” based on morphology (thin myelin, oligodendrocyte cytoplasm between myelin and axolemma) were excluded from the g-ratio analysis and counted as “remyelinating axons.” Quantifications were conducted by a blinded investigator.
Cell isolation for flow cytometry
Following transcardial perfusion with PBS, spinal cords and spleens were harvested and placed in cold HBSS. Samples were mechanically dissociated into single-cell suspensions through a 70 μm cell strainer and washed in HBSS. For leukocyte isolation from the spleen, suspensions were spun at 400 × g for 5 min, supernatants were removed, and red blood cells were lysed in 2 ml of lysis buffer (eBioscience) according to manufacturer instructions. Cells were then resuspended in flow cytometry buffer (FCB; eBioscience) and stained as described below. Leukocytes infiltrating into the spinal cord were isolated by negative selection of single-cell spinal cord suspensions with Myelin Removal Beads II in combination with LS columns, according to the manufacturer protocols (Miltenyi Biotec). Similar to the spleen cells, spinal cord cells were resuspended in FCB and stained as described below. The number of viable cells was determined by Trypan blue exclusion assay using a Bio-Rad TC20 automated cell counter.
Immunolabeling and flow cytometric analysis
Cells were resuspended in 100 μl of FCB, blocked with 2 μl of TruStainFcX (BioLegend) for 10 min at 4°C and stained for 30 min at 4°C with the following antibodies: APC-Cy7-anti-CD45 (1:200; #103116, BioLegend), FITC-anti-CD4 (1:200; catalog #11-0042, eBioscience), PerCP-Cy5.5-anti-CD8a (1:200; catalog #100734, eBioscience), PE-Cy7-anti-CD25 (1:200; catalog #25-0251-82, eBioscience), PE-anti-CD45R/B220 (1:200; catalog #103207, BioLegend), eFluor450-anti-CD11b (1:200; catalog #48-0112-82, eBioscience), APC-anti-O4 (1:100; catalog #130-095-891, Miltenyi Biotec), Alexa Fluor 488-anti-NG2 (1:200; catalog #AB5320A4, Millipore), or PE-anti-CD120b (TNFR2; 1:200; catalog #113406, BioLegend). Cell suspensions were then fixed and permeabilized (mouse Foxp3 staining kit, eBioscience) before intracellular staining with PE-anti-Foxp3 (1:100; catalog #12-5773-82, eBioscience). Cells were resuspended in 500 μl of FCB and analyzed with an LSRII flow cytometer (BD Biosciences) equipped with FACS-Diva 6.0 software (BD Biosciences).
RNA isolation and real-time PCR
Total RNA was extracted with TRIzol (Invitrogen) and reversed transcribed as previously described (Brambilla et al., 2011). Real-time PCR was performed in the Rotor-Gene 3000 Real Time Cycler (Corbett Life Science) as described previously (Brambilla et al., 2011). Primers for gene amplification are listed in Table 1. For miRNA amplification, following TRIzol extraction, total RNA was purified with the miRNeasy Micro Kit (Qiagen), then reverse transcribed with the Universal cDNA Synthesis Kit II (Exiqon). cDNA diluted 1:10 to 1:100 (depending on the abundance of the miRNA of interest) was amplified with the Power SYBR Green PCR Master Mix (Invitrogen) with primers designed for U6 (reference miRNA for normalization), miR-7a, and miR-138 (Exiqon), according to manufacturer recommendations. Quantitative PCR was performed on an ABI 7300 Real-Time PCR System (ThermoFisher Scientific). Relative miRNA expression was calculated using the ΔΔCt method (Livak and Schmittgen, 2001) and expressed as a percentage of WT oligodendrocyte precursor cell (OPC) miRNA expression.
Primary oligodendrocyte cultures
Cortices from postnatal day 4–6 pups were isolated and dissociated into single cell suspensions with Papain Neural Tissue Dissociation Kit (Miltenyi Biotec). PDGFRα+ OPCs were isolated by MACS separation using LS columns (Miltenyi Biotec) after incubation with PDGFRα magnetic microbeads. PDGFRα+ cells were cultured on poly-d-lysine/laminin-coated 24-well plates (40,000 cells/well) in OPC medium consisting of DMEM/F12/HEPES supplemented with 1% N2, 2% B27, 0.01% BSA, 7.5% NaHCO3, 1% penicillin/streptomycin, 10 ng/ml PDGF-AA, and 10 ng/ml FGF2. After 2 d in culture 100% of DAPI+ cells were positive for PDGFRα and 97.5% positive for Olig2. No cells stained for NeuN (neurons), GFAP (astrocytes), Iba1 (microglia), or PDGFRβ (fibroblasts). Cells were maintained in OPC medium for 4 d. At day 4, cells were either fixed/collected or switched to oligodendrocyte (OL) differentiation medium consisting of DMEM/F12/HEPES supplemented with 1% N2, 2% B27, 0.01% BSA, 7.5% NaHCO3, 0.5% gentamycin, 10 ng/ml CNTF, and 40 ng/ml T3. Cells were differentiated to premyelinating OLs for 4 d then fixed or collected for RNA extraction. OLs were identified by O1 labeling of live cells with hybridoma culture supernatant or MPB staining (rat, 1:500; catalog #MAB386, Millipore). Immunoreactivity was visualized with secondary species-specific fluorescent antibodies (1:750; Alexa Fluor-594 and Alexa Fluor-488, Invitrogen).
Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling staining
Apoptotic cell death was detected with the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay (Millipore) according to manufacturer protocols. Biotin-labeled fragmented DNA was visualized with strepdavidin-conjugated Alexa Fluor-647 (1:2000; catalog #S-32357, Invitrogen). OPCs and OLs were identified by double labeling with anti-PDGFRα (rabbit; 1:200; catalog #SC-338, Santa Cruz Biotechnology) and anti-APC (CC1 clone; mouse; catalog #OP80, 1:500, Millipore), respectively, followed by Alexa Fluor-488 goat anti-mouse secondary antibody (1:750; catalog #A11001, Invitrogen). For each well, five images were acquired at a 20× magnification, and PDGFRα+TUNEL+ and CC1+TUNEL+ cells were counted blindly.
Oligodendrocyte proliferation and differentiation by 5-ethynyl-2-deoxyuridine incorporation
OPCs were incubated with 10 μm 5-ethynyl-2-deoxyuridine (EdU) for 8 h at 37°C, either fixed with 4% PFA for 15 min at room temperature or switched to OL medium, and differentiated for 4 d. EdU incorporation was detected with the Click-iT EdU Alexa Fluor-594 Imaging Kit (Invitrogen) according to manufacturer protocols. OPC proliferation was assessed as the ratio of PDGFRα+EdU+ cells to total PDGFRα+ cells. OL differentiation was assessed as the ratio of CC1+EdU+ cells to total CC1+ cells. Each condition was run in four replicates, and for each replicate the cell number was determined as the average of five images randomly collected. All images were recorded by a blinded investigator. Data are the average ± SEM of three independent experiments.
Spinal cords were harvested in cold HBSS and dissociated with Papain Neural Dissociation Kit (Miltenyi Biotec). Single-cell suspensions were cleared from myelin by incubation with Myelin Removal Beads II followed by negative selection with LS columns (Miltenyi Biotec). To select for cells of the oligodendrocyte lineage, suspensions were incubated with 10 μl of anti-PDGFRα plus 10 μl of anti-O4 magnetic microbeads (Miltenyi Biotec) for 15 min at 4°C, then positively selected with LS columns (Miltenyi Biotec). Cell viability and oligodendrocyte enrichment were verified by flow cytometry using APC-anti-O4 (1:100; catalog #130-095-891, Miltenyi Biotec) and PE-anti-PDGFRα (1:100; catalog #SC-338PE, Santa Cruz Biotechnology) antibodies, and the fraction of PDGFRα+ and O4+ cells combined was assessed to be consistently >40% of the total number of viable cells. Total RNA was isolated with a miRNeasy Kit (Qiagen), and microRNA expression was assayed with the microRNA Ready-to-Use PCR Array (Mouse&Rat version 2.R; Exiqon). Arrays were run on a LightCycler 480 real-time PCR machine (Roche) at the Oncogenomics Core Facility of the University of Miami. Data analysis was performed using the GenEx software (Multid Analyses AB). For each comparison, output files from the LightCycler were uploaded to the software and an interplate calibration was performed. Normalization was performed using the global mean method (Mestdagh et al., 2009). Statistical significance was assessed by an unpaired two-tailed t test.
In situ hybridization
Thirty-micrometer-thick isopropanol-fixed spinal cord sections were subjected to standard in situ hybridization, as previously described, using an alkaline phosphatase-labeled LNA probe recognizing mouse miR-7 (miR-7 probe: ACAACAAAATCACTAGTCTTCCA; DNA Technology A/S; Hansen et al., 2013). Sections were rinsed 15 min in TBS plus 1% Triton X-100 and incubated with anti-PDGFRα antibody (1:250; catalog #SC-338, Santa Cruz Biotechnology) in TBS plus 10% bovine serum overnight, then incubated with Alexa Fluor-546 goat anti-rabbit IgG (1:200; Invitrogen) for 1 h. Sections were mounted with Aquatex (EMD Millipore). Expression of miR-7 was visualized with bright-field imaging. Colocalization with PDGFRα immunostaining was determined after conversion of the signal to green pseudocolor using Adobe Photoshop software (Lambertsen et al., 2009). The number of miR-7a+ cells was quantified in the intact white matter on spinal cord serial sections at 300 μm intervals using ImageJ by an investigator blinded to the genotype. Four to eight sections/mouse were analyzed, and numbers were normalized to the area of intact white matter.
Statistical analysis of the clinical course of EAE was performed with the Mann–Whitney U test. Histological data, flow cytometry assessments, and real-time PCR were analyzed by either one-way or two-way ANOVA followed by Bonferroni's or Tukey's test for multiple comparisons. In the case of single comparisons, the Student's t test was applied. The g-ratio data were analyzed by linear regression, and slope and elevation parameters were used to compare groups. p values ≤0.05 were considered to be statistically significant. Data were expressed as the average of multiple determinations ± SEM. Statistical analyses were conducted with GraphPad Prism software.
Generation and characterization of CNP-cre:TNFR2fl/fl mice
To assess the pathophysiological function of oligodendroglial TNFR2, we established CNP-cre:TNFR2fl/fl conditional KOs where TNFR2 is ablated from OPCs and OLs. CNP-cre+/− mice (Lappe-Siefke et al., 2003) were crossed with TNFR2fl/fl mutants obtained by breeding C57BL/6NTac-Tnfrsf1btm1a(EUCOMM)Wtsi/Ics mice with an Flp1 deleter line to remove the FRT-flanked neomycin cassette (Fig. 1A,B). Recombination efficiency was evaluated by crossing CNP-cre+/− mice with Rosa26-stop-EYFP reporter mice and determined to be ∼60% in OPCs and close to 100% in OLs (Fig. 1C), indicating that CNP-cre mice are suitable for TNFR2 ablation throughout the lineage. TNFR2 expression in NG2+ OPCs and O4+ OLs was found to be significantly reduced in CNP-cre:TNFR2fl/fl mice by ∼25% and 40%, respectively (Fig. 1D). Stereological counting of PDGFRα+TNFR2+ OPCs and CC1+TNFR2+ OLs yielded similar results (Fig. 1E,F). In addition, TNFR2 gene expression in the spinal cord was downregulated compared with TNFR2fl/fl, WT, and CNP-cre+/− mice (Fig. 1G). Importantly, oligodendroglial TNFR2 ablation did not result in changes in the number of NG2+PDGFRα+ OPCs (Fig. 1H) or CC1+ OLs (Fig. 1I,J), suggesting that TNFR2 is not implicated in the regulation of oligodendrocyte survival and/or maturation during development. To further investigate whether TNFR2 ablation might interfere with normal oligodendrocyte function, we quantified the expression of genes involved in inflammation and myelination in the spinal cord and found no difference between CNP-cre:TNFR2fl/fl mice and any of the control groups (Fig. 2A). Finally, we assessed spontaneous activity and locomotor function with the rotarod test (Fig. 2B,C) and open field test (Fig. 2D–I), where CNP-cre:TNFR2fl/fl mice performed equally to controls. Typical and anxiety-related behaviors were not altered as well (Fig. 2E–I). Collectively, these data indicate that CNP-cre:TNFR2fl/fl mice are a suitable model to study oligodendroglial TNFR2 function in vivo.
Ablation of oligodendroglial TNFR2 results in EAE exacerbation
To assess whether oligodendroglial TNFR2 mediates tmTNF protective function in neuroimmune disease, we induced EAE with MOG35–55 peptide (Brambilla et al., 2014). CNP-cre:TNFR2fl/fl mice showed a significantly worse clinical outcome than TNFR2fl/fl controls, with earlier onset and exacerbated chronic disease (Fig. 3A, Table 2). The administration of the solTNF inhibitor XPro1595, which improved EAE in TNFR2fl/fl controls in line with our previous studies (Brambilla et al., 2011), did not show any benefit in CNP-cre:TNFR2fl/fl mice, confirming that the therapeutic outcome of solTNF inhibition is dependent on a functional TNFR2 in oligodendrocytes. Immune cell infiltration into the spinal cord mirrored the clinical course of the disease. At peak EAE (19 dpi), CNP-cre:TNFR2fl/fl mice had a significantly higher percentage of infiltrating CD4+ and CD8+ T cells, as well as a higher percentage of regulatory T cells (Fig. 3B). By 25 dpi, when disease scores were almost overlapping, infiltrating cells were comparable between genotypes (Fig. 3C). In the spleen, no differences were found in naive conditions or after EAE (Fig. 3D–F). The acute phase of EAE was characterized by the upregulation of proinflammatory mediators like TNF, IL-1β, IFNγ, and C-X-C ligand-10 (CXCL10; Fig. 3G), but no differences were found between CNP-cre:TNFR2fl/fl and TNFR2fl/fl mice, likely due to the similar EAE severity at this stage of disease. Interestingly, we found changes in the expression of TNF receptors. TNFR2 was upregulated in TNFR2fl/fl mice after EAE but not in CNP-cre:TNFR2fl/fl mice, which, as anticipated, showed significant reduction of TNFR2 in naive as well as disease conditions. TNFR1 was upregulated after EAE in both genotypes, but with a higher increment in CNP-cre:TNFR2fl/fl mice. This could be due to the increased infiltration of peripheral immune cells, which are known to highly express TNFR1 (Vielhauer et al., 2005; Liu and Tang, 2014).
Recent reports (Hagemeyer et al., 2012; Wieser et al., 2013) have shown that CNP-cre−/+ mice display white matter abnormalities and axonal degeneration following aging or mild CNS trauma. This is due to the CNPase loss of function resulting from CNP-cre−/+ mice having only one allele of the endogenous Cnp1 gene (Lappe-Siefke et al., 2003), and this has highlighted the key role of CNPase in myelin stability. Therefore, to exclude that Cnp1 haploinsuffiency of the CNP-cre−/+ drivers could be responsible, at least in part, for the worsening of the clinical outcome in CNP-cre:TNFR2fl/fl mice, we performed a control experiment to assess the clinical course of EAE in CNP-cre+/− versus WT mice. CNP-cre+/− mice developed EAE similar to controls (Fig. 3H) and showed comparable axonal damage during chronic disease (Fig. 3I). This indicates that CNPase loss of function is not a factor in EAE exacerbation in CNP-cre:TNFR2fl/fl mice, which depends only upon TNFR2 ablation.
Ablation of oligodendroglial TNFR2 increases axonal loss after EAE
To assess whether worsening of the clinical outcome in CNP-cre:TNFR2fl/fl mice could be associated with more severe axonal damage, we quantified the number of intact and degenerated axons in the spinal cord in naive conditions and chronic EAE (Fig. 4A). No differences were found in naive mice, and with chronic EAE both groups displayed widespread axonal loss and accumulation of degenerated axons (Fig. 4A, black arrows). This damage was further exacerbated in CNP-cre:TNFR2fl/fl mice, who showed a significant reduction in myelinated axons (Fig. 4B) and an increase in degenerated axons (Fig. 4C). This suggests that oligodendroglial TNFR2 plays a neuroprotective function in pathological conditions.
Ablation of oligodendroglial TNFR2 increases myelin pathology and reduces remyelination after EAE
Next, we investigated whether the increased axonal damage in CNP-cre:TNFR2fl/fl mice was associated with worse oligodendrocyte and myelin pathology. We found a significant reduction in the number of PDGFRα+ OPCs in CNP-cre:TNFR2fl/fl mice after EAE (Fig. 4D,E). CC1+ OLs were also diminished compared with naive mice (Fig. 4F,G), but the loss was significantly higher in CNP-cre:TNFR2fl/fl mice (Fig. 4G), suggesting that oligodendroglial TNFR2 may play a role in the survival of existing OLs and/or differentiation of OPCs into new OLs.
Myelin integrity in the spinal cord white matter was examined by EM. Morphologically, naive mice did not show myelin abnormalities, but following EAE both genotypes displayed extensive damage, with unraveling of the myelin sheaths, loss of adaxonal contact, and complete demyelination in severe cases (Fig. 5A). To measure myelin thickness, we assessed the g-ratio (axon diameter/fiber diameter) of individual axons. We plotted the g-ratio against the corresponding axon diameter and did not find any difference in naive conditions by linear regression analysis (line slope and elevation intercept on the y-axis were comparable; Fig. 5B). However, following EAE, CNP-cre:TNFR2fl/fl mice showed significantly and consistently lower g-ratios compared with TNFR2fl/fl controls, shown by reduced elevation of the linear regression (CNP-cre:TNFR2fl/fl = 0.575 ± 0.007 vs TNFR2fl/fl = 0.615 ± 0.005; p ≤ 0.0001, Student's t test) without change in slope (Fig. 5C). This was evident also by grouping axons by diameter size, with the g-ratio significantly reduced after EAE in CNP-cre:TNFR2fl/fl mice, except for axons of >2 μm (Fig. 5D). This indicates that CNP-cre:TNFR2fl/fl mice have consistently wider myelin rings compared with controls. It is important to note that axons identified morphologically as undergoing remyelination (thin compact myelin, often with interposition of oligodendrocyte cytoplasm between axon and the myelin ring) were not included in the g-ratio analysis, but were counted separately for more accurate assessment of remyelination. Remyelinating axons (Fig. 5E) were reduced in CNP-cre:TNFR2fl/fl mice compared with TNFR2fl/fl controls (Fig. 5F), suggesting that TNFR2 in oligodendrocytes plays a role, directly or indirectly, in sustaining the remyelination process.
TNFR2 regulates oligodendrocyte differentiation
To address whether oligodendroglial TNFR2 is involved in survival, proliferation, or differentiation of OPCs and OLs, we established an in vitro system of WT and TNFR2−/− OPC/OL cultures. To assess TNFR2-dependent cell survival, we counted PDGFRα+TUNEL+ OPCs and CC1+TUNEL+ OLs, and found no difference between WT and TNFR2−/− cells (Fig. 6A,C). The ratio of PDGFRα+TUNEL+ cells to the total number of PDGFRα+ OPCs or CC1+TUNEL+ cells to the total number of CC1+ OLs did not vary (Fig. 6B,D), demonstrating that TNFR2 is not required for OPC or OL survival in vitro. Nevertheless, the total number of CC1+ OLs was significantly reduced in TNFR2−/− cells (Fig. 6C), suggesting that TNFR2 could be modulating OPC proliferation, differentiation, or both. To assess proliferation, OPCs were incubated with EdU and fixed 8 h later. We found no difference between WT and TNFR2−/− OPCs in the number of PDGFRα+EdU+ actively proliferating cells (Fig. 6E), and the ratios of PDGFRα+EdU+ to total PDGFRα+ cells were similar (Fig. 6F). To assess OPC differentiation, 8 h after the addition of EdU cells were switched to differentiation medium and fixed 4 d later. We counted CC1+EdU+ OLs and found that a reduced number of EdU-incorporating OPCs differentiated into OLs in TNFR2−/− cultures compared with WT (Fig. 6E). This was also shown by the lower ratio of CC1+EdU+ cells to PDGFRα+EdU+ cells (Fig. 6G), indicating that TNFR2 is important for OPC differentiation but not proliferation. The reduced differentiation capability of cells lacking TNFR2 was further demonstrated by immunocytochemistry in differentiated cultures (Fig. 6H). WT cells showed highly differentiated flat morphology, with diffused O1 and MBP immunoreactivity. TNFR2−/− cells appeared immature, with thin elongated processes, small cell body, and reduced MBP immunoreactivity. Impaired differentiation was also demonstrated by gene expression analysis (Fig. 6I), with PDGFRα significantly higher in TNFR2−/− OLs and not different from undifferentiated OPCs, MBP drastically reduced in TNFR2−/− OLs, and notch1, which acts as a brake to OPC differentiation (Juryńczyk and Selmaj, 2010), elevated in TNFR2−/− OPCs. Collectively, these data demonstrate that TNFR2 drives OPC differentiation but does not regulate OPC survival or proliferation.
Ablation of oligodendroglial TNFR2 dysregulates miRNA expression
To address the mechanisms of TNFR2-dependent regulation of OPC differentiation, we investigated changes in miRNA expression in the spinal cord of TNFR2fl/fl and CNP-cre:TNFR2fl/fl mice in naive and 25 dpi EAE conditions. Since miRNAs are known to regulate oligodendrocyte differentiation (Dugas et al., 2010; Zhao et al., 2010; He et al., 2012), and can modulate or be modulated by TNF signaling (Suárez et al., 2010; Chen et al., 2014), we quantified miRNA expression in oligodendrocyte-enriched spinal cord suspensions. In naive mice, we found differential expression of 29 miRNAs (Table 3). Most notably, miR-7a, which has been implicated in arresting oligodendrocyte differentiation (Zhao et al., 2012), was increased by 3.58-fold in CNP-cre:TNFR2fl/fl mice compared with TNFR2fl/fl mice. After EAE, we saw differential expression of 10 miRNAs in CNP-cre:TNFR2fl/fl mice compared with TNFR2fl/fl controls (Table 3). Interestingly, inflammatory miR-326, reportedly associated with MS and EAE pathogenesis by promoting Th17 differentiation (Du et al., 2009; Honardoost et al., 2014), was upregulated in CNP-cre:TNFR2fl/fl mice.
As anticipated, we found a large number of miRNAs altered as a consequence of EAE within each genotype. Nevertheless, when we focused on those involved in OPC differentiation and myelination (Table 4), we found different expression patterns in TNFR2fl/fl and CNP-cre:TNFR2fl/fl mice. Two members of the miR-219 and miR-138 clusters, and miR-338–5p, all required for oligodendrocyte differentiation (Dugas et al., 2010; Zhao et al., 2010; de Faria et al., 2012; Pusic and Kraig, 2015), were downregulated in CNP-cre:TNFR2fl/fl mice, but not in TNFR2fl/fl controls, suggesting that oligodendroglial TNFR2 may be regulating OPC differentiation in disease conditions via modulation of these molecules. Similarly, miR-7a changed only in CNP-cre:TNFR2fl/fl mice with significant downregulation after EAE. Since miR-7a showed the highest differential expression in naive conditions (Table 3), we further investigated whether it played a direct role in the protective function of oligodendroglial TNFR2 in EAE. We quantified miR-7a expression in vivo by in situ hybridization in naive and 25 dpi EAE conditions, when the peak of the inflammatory response is over and the repair process is underway. In agreement with previous studies localizing miR-7a in the CNS mainly in OPCs (Zhao et al., 2012), in the spinal cord white matter we found miR-7a to be almost exclusively expressed in PDGFRα+ OPCs (Fig. 7A). The number of miR-7a+ cells in the intact white matter was significantly increased in CNP-cre:TNFR2fl/fl mice compared with TNFR2fl/fl controls in naive conditions (Fig. 7B), mirroring the PCR array data (Table 3). However, at 25 dpi EAE expression reversed, with a significant reduction in the number miR-7a+ cells in CNP-cre:TNFR2fl/fl mice (Fig. 7B). To better understand whether TNFR2 could be directly modulating miR-7a within the oligodendrocyte population during differentiation, we measured miR-7a expression by real-time PCR in OPCs and OLs from WT and TNFR2−/− mice. Unlike the EAE condition in vivo where oligodendrocytes are exposed to a highly proinflammatory environment, in vitro we tested miR-7a expression in nonstimulated conditions. In WT cultures, miR-7a expression dropped as cells differentiated to the OL stage (Fig. 7C). However, in the absence of TNFR2, miR-7a stayed elevated at the OL stage (Fig. 7C) and coincided with the failure of cell maturation (Fig. 6H,I). In line with the report by Zhao et al. (2012) showing that blockade of miR-7a in differentiating neural progenitors leads to reduced OL numbers, our data suggest that TNFR2 may drive OPC differentiation at least in part by repressing miR-7a function.
We also tested in vitro the expression of miR-138, which we found differentially regulated in our microRNA screening after EAE (Table 3). miR-138 was significantly higher in TNFR2−/− OPCs compared with WT cells (Fig. 6C). In differentiated OLs, expression was increased with no difference between WT and TNFR2−/− cells (Fig. 7C). This suggests that miR-138, an important signal in oligodendrocyte differentiation, is likely not regulated by TNFR2.
Using new oligodendrocyte-specific TNFR2 conditional KO mice in combination with the solTNF inhibitor XPro1595, we demonstrate that TNFR2 expressed in the oligodendrocyte lineage contributes to tmTNF-mediated beneficial effects in EAE, providing the first direct in vivo evidence that oligodendroglial TNFR2 is protective in neuroimmune disease. We previously demonstrated that tmTNF signaling is necessary for functional recovery, axon preservation, and remyelination in EAE (Brambilla et al., 2011), and here we address the molecular mechanisms of this effect by showing they depend, at least in part, on direct activation of TNFR2 in oligodendrocytes. Aside from a study by Arnett et al. (2001) in whole-animal TNFR2−/− mice suggesting a protective role for TNFR2 in demyelination, and a study by Maier et al. (2013) reporting that the activation of TNFR2 in OPCs is protective against oxidative stress, the function of oligodendroglial TNFR2 is largely unknown. With the generation of CNP-cre:TNFR2fl/fl mice, we were able to directly investigate the biological function of oligodendroglial TNFR2 both in normal and pathological conditions. Under naive conditions, ablation of TNFR2 did not alter oligodendrocyte cell number or impair locomotor function and spontaneous activity, indicating that TNFR2 is not implicated in the regulation of oligodendroglial function during development. After EAE, however, TNFR2 ablation resulted in a worsening of the clinical outcome that was not rescued by treatment with XPro1595. This indicates that tmTNF-mediated beneficial effects require the presence of TNFR2 in oligodendrocytes. It also underscores that worsening of the EAE outcome in CNP-cre:TNFR2fl/fl mice cannot simply be attributed to an “unmasking” of TNFR1 detrimental function. If that was the case, XPro1595 would be effective in protecting mice from EAE, because it selectively inhibits solTNF, which activates TNFR1.
In CNP-cre:TNFR2fl/fl mice, EAE symptoms manifested earlier and were more severe than in control mice at chronic disease. This suggests that oligodendroglial TNFR2 likely contributes to protection from EAE via multiple mechanisms: it may serve as anti-inflammatory signal, hence affecting the onset of the disease; and may provide signals benefitting axon and myelin integrity, thereby affecting the chronic phase of the pathology. The notion that oligodendroglial TNFR2 may be implicated in suppressing the inflammatory response is supported by two lines of evidence: first, CNP-cre:TNFR2fl/fl mice showed a higher percentage of infiltrating CD4+ and CD8+ T cells at peak disease; and, second, proinflammatory miRNAs were found to be upregulated in CNP-cre:TNFR2fl/fl mice at the acute stage of disease. The most notable example is miR-326, which has been linked to MS and EAE pathogenesis as a promoter of Th17 differentiation (Du et al., 2009; Honardoost et al., 2014). miR-326 has been found upregulated in the blood of patients undergoing relapse (Honardoost et al., 2014) and diminished after natalizumab treatment (Ingwersen et al., 2015). Whether the anti-inflammatory effect of oligodendroglial TNFR2 occurs directly in oligodendrocytes or indirectly via other cell populations remains to be seen. Regardless, our data highlight that oligodendrocytes have functions that go beyond myelination, repair, and metabolic support of neurons, which is in tune with the roles of the other glial cell populations. TNFR2 activation in microglia has been shown to induce the production of the neuroprotective molecules granulocyte colony-stimulating factor, adrenomedullin, and IL-10 (Veroni et al., 2010). Hence, we can speculate that a similar mechanism may be at play in oligodendrocytes, and the reduced production of anti-inflammatory signals in CNP-cre:TNFR2fl/fl mice may explain the greater influx of encephalitogenic T cells into the CNS. Ultimately, a better understanding of the role of oligodendroglial TNFR2 in inflammation is important to have a complete picture of the function of this receptor in neuroimmune disease, and this is the focus of ongoing studies in our laboratory.
In addition to an anti-inflammatory function, our data point to an effect of oligodendroglial TNFR2 in neuroprotection, myelin repair, and remyelination. Indeed, the ablation of TNFR2 in CNP-cre:TNFR2fl/fl mice resulted in increased axonal loss and myelin damage, as well as reduced remyelination. The reduced g-ratios measured in CNP-cre:TNFR2fl/fl mice indicate the presence of thicker myelin rings. Because it is unlikely that this depends on oligodendrocytes producing more myelin and wrapping more layers around the axons, it is plausible that thicker rings are the consequence of a loss of myelin compaction and/or axonal atrophy, suggesting that TNFR2 plays a role in the maintenance of myelin and axon integrity in disease conditions. In the absence of oligodendroglial TNFR2, the pool of OPCs was drastically depleted following EAE, and in vitro experiments demonstrated that TNFR2 is required for OPC differentiation. This is significant because efficient OPC differentiation is the prerequisite for repair in demyelinating pathologies (Kremer et al., 2015). Failure of this process prevents remyelination, leading to axonal damage and ultimately to neuronal loss, which is the cause of permanent disability in MS. While reports have shown that TNFR2 in astrocytes (Patel et al., 2012; Fischer et al., 2014) may activate cascades that indirectly stimulate OPC differentiation, here we show for the first time that direct TNFR2 activation in OPCs is crucial in promoting differentiation, thereby sustaining the remyelination process. We did not observe a developmental impairment of OPC differentiation in vivo, because oligodendrocytes numbers were comparable in naive TNFR2fl/fl and CNP-cre:TNFR2fl/fl mice. This apparent contradiction with our in vitro data can be reconciled in two ways. First, CNP-cre mice allow for TNFR2 ablation in <40% of OPCs, and this may not translate into a developmental phenotype. Better suited to address OPC differentiation in vivo are PDGFRα-creER drivers, where recombination is highly efficient and specific in OPCs. Experiments with this mouse line are currently ongoing in the laboratory. Second, unlike the in vitro model where purified OPCs do not receive inputs from other cell types, OPC differentiation in vivo is controlled by the concerted action of numerous environmental signals converging on the OPCs. Therefore, it is likely that other mechanisms compensate for TNFR2 absence, allowing for normal OPC differentiation.
The cascades initiated by TNFR2 to drive differentiation include the modulation of miRNAs, such as miR-7a. It was recently reported that the overexpression of miR-7a in OPCs promotes proliferation and inhibits differentiation into a mature OL phenotype (Zhao et al., 2012), and overexpression in retinal glial precursors reduces the differentiation of Müller glia (Baba et al., 2015). Additionally, in adult rats it has been shown that miR-7a expression is downregulated in mature OLs compared with OPCs (Lau et al., 2008). Our expression data, both from PCR array and in situ hybridization, show that in the absence of oligodendroglial TNFR2 miR-7a expression in the spinal cord white matter is elevated under naive conditions. Since we found almost exclusive expression of miR-7a in PDGFRα+ OPCs in the white matter, in agreement with previous reports (Zhao et al., 2012), this suggests a direct regulation of miR-7a expression in OPCs by TNFR2. Furthermore, because we showed that OPCs and OLs do not change in numbers in naive conditions when TNFR2 is ablated, this suggests that TNFR2-dependent modulation of miR-7a in oligodendrocytes does not play a role during development. We could speculate that this mechanism might be important only after injury to the CNS, such as in EAE, when TNFR2 expression is upregulated in all glial populations, including oligodendrocytes. After EAE, the lack of oligodendroglial TNFR2 results in reduced numbers of miR-7a-expressing cells in the white matter (mostly OPCs). This may depend on the higher OPC loss seen in CNP-creTNFR2fl/fl mice, but it may also be an adaptive response in an attempt to boost OPC differentiation to sustain the repair and remyelination process. In vitro, in noninflammatory conditions, we showed that the drop in miR-7a expression occurring when OPCs transition to OLs does not take place in the absence of TNFR2, demonstrating that TNFR2 directly regulates miR-7a. Nevertheless, we cannot exclude that oligodendroglial TNFR2 might also be modulating miR-7a expression indirectly.
In addition to miR-7a, others known to regulate oligodendrocyte differentiation were found to be dysregulated as a consequence of TNFR2 ablation, specifically members of the miR-219 and miR-138 clusters, as well as miR-338–5p. All of these miRNAs are highly expressed in the spinal cord, and some are required for normal oligodendrocyte differentiation and myelination in both rodents and humans (Dugas et al., 2010; Zhao et al., 2010; de Faria et al., 2012). We found these molecules to be highly downregulated after EAE in CNP-cre:TNFR2fl/fl mice, but not in TNFR2fl/fl mice, providing yet another indication that TNFR2 is necessary for activating OPC differentiation programs without which remyelination cannot effectively take place.
In summary, our study provides the first direct evidence that TNFR2 is an important signal for oligodendrocyte differentiation. Following activation by tmTNF, TNFR2 initiates specific pathways, which include miRNA modulation, that drive oligodendrocytes into a reparative mode contributing to remyelination following disease, thereby preventing axonal loss and disability. This identifies TNFR2 as a viable new therapeutic target in neuroimmune disease. Selective TNFR2 agonists and selective solTNF inhibitors, which skew the balance toward TNFR2 activation, could be used separately or in combination to promote myelin repair. This may represent a new, much-needed pharmacological tool for the primary progressive form of MS where disability is directly dependent on irreparable axonal damage associated with demyelination and for which no effective treatments are available.
This work was supported by FISM (Fondazione Italiana Sclerosi Multipla) Grant 2012/R/2 (R.B.); National Institutes of Health/National Institute of Neurological Disorders and Stroke Grant NS-084303-01A1 (R.B.); the Miami Project To Cure Paralysis (R.B.); the Danish Multiple Sclerosis Society Grant R431-A29647-B20593 (P.M.M., K.L.L., R.B.); Fonden til Lægevidenskabens Fremme (P.M.M.); and Fonden for Neurologisk Forskning (P.M.M.). We thank Margaret Bates and Vania Almeida at the Electron Microscopy Core Facility of The Miami Project To Cure Paralysis for assistance in electron microscope imaging. We also thank Mehran Taherian for skillful technical assistance and Dr. Paula Monje for providing the anti-O1 hybridoma supernatant. In addition, we thank Dr. Klaus Nave (Max Planck Institute of Experimental Medicine, Göttingen, Germany) for providing the CNP-cre+/− mouse line.
D.E.S. is an employee of Xencor and also holds stocks and stock options in the company. All other authors have no financial conflicts of interest to declare.
- Correspondence should be addressed to Dr. Roberta Brambilla, The Miami Project To Cure Paralysis, University of Miami Miller School of Medicine, 1095 NW 14th Terrace, Miami, FL 33136.


text:
Howard Douglas : biography
General Sir Howard Douglas, 3rd Baronet, GCB, GCMG, FRS (23 January 1776 – 9 November 1861) was a British military officer born in Gosport, England, the younger son of Admiral Sir Charles Douglas, and a descendant of the Earls of Morton. He was a British general and colonial administrator.
Later career in Europe
On his return to Europe he was employed in various missions, and he published about this time Naval Evolutions, a controversial work dealing with the question of breaking the line (London, 1832). From 1835 to 1840 Douglas, now a GCMG, was Lord High Commissioner of the Ionian Islands, where, amongst other reforms, he introduced a new code of laws. In 1837 he became a Lieutenant-General, in 1840 a KCB, in 1841 a civil GCB, and in 1851 a General. From 1842 to 1847 Douglas sat as a Conservative Member of Parliament (MP) for Liverpool, where he took a prominent part in debates on military and naval matters and on the corn laws. He was frequently consulted on important military questions. His later works included Observations on the Modern System of Fortification, (London, 1859), and Naval Warfare Under Steam (London, 1858 and 1860). He died in Tunbridge Wells. Sir Howard Douglas was an FRS, one of the founders of the RGS, and an honorary DCL of Oxford University. Shortly before his death he declined the offer of a military GCB.
Early writings and promotion to Major-General
In 1816 appeared his Essay on the Principles and Construction of Military Bridges (subsequent editions 1832, 1853); in 1819, Observations on the Motives, Errors and Tendency of M. Carnots System of Defence, and in the following year his Treatise on Naval Gunnery (of which numerous editions and translations appeared up to the general introduction of rifled ordnance). In 1821 he was promoted Major-General. Douglas’s criticisms of Carnot led to an important experiment being carried out at Woolwich in 1822, and his Naval Gunnery became a standard text-book, and indeed first drew attention to the subject of which it treated.
In 1795 he was shipwrecked while in charge of a draft for Canada, and lived with his men for a whole winter on the Labrador coast. Soon after his return to England in 1799 he was made a Captain-Lieutenant. In his regimental service during the next few years, he was attached to all branches of the artillery in succession, becoming Captain in 1804, after which he was placed on half-pay to serve at the Royal Military College, then located at High Wycombe. He taught military strategy and was an authority on military and naval engineering. He served intermittently as commandant of the senior department and as inspector general of instructions at the Royal Military College. In 1804, Douglas was appointed to a majority in the York Rangers, a corps immediately afterwards reduced. He remained on the roll of its officers until promoted Major-General. The senior department of the RMC at High Wycombe, of which he was in charge, was the forerunner of the Staff College. Douglas was promoted brevet Lieutenant-Colonel in 1806. He served in 1808-1809 in the Peninsular War. He was present at the Battle of Corunna, after which he took part in the Walcheren expedition.
On the death of his half-brother, Vice-Admiral Sir William Douglas in 1809, he succeeded to the baronetcy. In 1812 he was employed in special missions in the north of Spain, and took part in numerous minor operations in this region, but he was soon recalled, the Home Government deeming his services indispensable to the Royal Military College. He became brevet Colonel in 1814 and CB in 1815. He became a fellow of the Royal Society on 25 January 1816.
In 1797, while in Quebec City, Douglas fathered a daughter, Margaret (or Marguerite), but did not marry the mother, Catherine Normandeau. In 1799, he returned to England, and in July of that year he married Anne Dundas, daughter of James Dundas. They had ten children, Major Charles Douglas, James Dundas Douglas, Howard Douglas, General Sir Robert Percy Douglas, 4th Baronet of Carr, Reverend William Frederick Douglas, Ann Douglas, Christina Douglas, Lucy Douglas, Sarah Mary Harcourt Douglas, and Mary Douglas.
Lieutenant-Governor of New Brunswick
Sir Howard Douglas became Governor of New Brunswick (1823–31). He had to deal with the Maine boundary dispute with the United States of 1828. He also founded Fredericton College (King’s College), now known as the University of New Brunswick, of which he was the first Chancellor. He was governor during the Miramichi fire of 1825, and his actions during that crisis increased his popularity with the people of the province. He secured a charter for King’s College at Fredericton (later the University of New Brunswick).
Following the death of his mother, Sarah Wood Douglas, in 1779, Howard was raised by his aunt, Helena Baillie, near Edinburgh. As a boy, he wanted to join the Royal Navy and follow in the footsteps of his father and older brother. His father agreed to take him to sea when he was thirteen years of age, but Sir Charles died of apoplexy while in Edinburgh just after he arrived to collect Howard in 1789. Howard’s guardians thought it better that he serve in the Army instead, and he entered the Royal Military Academy, Woolwich, in 1790. He was commissioned Second Lieutenant in the Royal Artillery in 1794, becoming Lieutenant a few months later.

domain: history


text:
The first tally of damage claims arising from the Exxon Valdez oil spill comes to $59 billion, with 11,105 claims filed, according to court documents Thursday.
A flood of new claims has more than doubled the number of damage claims that have been filed as a result of the 11 million-gallon Exxon Valdez tanker spill in Alaska's Prince William Sound, America's worst spill."There is some air of unreality to the dollar amount," said Stephen Hut Jr., a lawyer representing the Trans-Alaska Pipeline Liability Fund, which is sifting through mounds of complaints at the request of the federal court.
U.S. District Judge H. Russel Holland had instructed everyone suing Exxon to submit damage claims to the pipeline liability fund for possible payout before the court considered the numerous suits.
The many suits mention no dollar amounts though it was widely believed that damage claims went into the billions. But claims filed with the pipeline liability fund were required to state a dollar amount of damages, thus offering the first documented accounting of damages by those seeking spill damages.
However, when Congress established the pipeline liability fund in 1973 - assessing oil company owners of the trans-Alaska oil pipeline 5 cents per barrel to create a $100 million fund to pay for spill damages - no one envisioned a spill as costly as the March 24, 1989, wreck of the Exxon Valdez has turned out to be.
"The amount claimed is far in excess of that fund," Hut noted. "We cannot pay until we know what everyone is entitled to."
Lawyers for the pipeline liability fund summarized all Exxon Valdez damage claims in a report filed in federal court Monday but they only became public three days later because of the huge volume of new documents submitted to the court in the Exxon case.
"The fund was deluged with claims" just before the March 25 deadline, doubling the number of claims that had been filed as of March 1, the fund report said.
Joining the crowd seeking damages were the state of Alaska, the U.S. government and five oil companies - including Exxon itself, which said it should be reimbursed $1.3 billion of the $2 billion it spent to clean up the spill.

domain: law


text:
|Code Enforcement Contact|
1600 W. Beverly Blvd.
Montebello, CA 90640
Vacant and Abandoned Properties
As of September of 2008 there were over 200 properties within the City of Montebello is some stage of foreclosure. People are abandoning properties they can no longer afford to keep and real estate market is flooded with more available properties than buyers. Many of these properties are in a state of ownership limbo: the legal owner abandons the property and the bank (or another potential new owner) does not have legal control until the foreclosure auction.
The vacant and abandoned properties are becoming an issue for both Code Enforcement and our community in general. Many of these properties are not being maintained causing neighborhood blight. Some of these abandoned properties are being used by squatters for homes, some by local youth for parties and some by criminals for illegal activities.
In addition to the problems outlined above, these properties can pose health and safety hazards to a neighborhood. For example, properties that have swimming pools or other water features that are left unmaintained breed mosquitoes which can lead to the spread of the West Nile Virus. Some of these abandoned properties become fire hazards presenting a threat to surrounding properties.
To report a vacant and abandoned property with maintenance issues please contact Code Enforcement at (323) 887-1490.


Gerard Butler & John Mayer: Birthday Duet!
The 41-year-old Scottish actor rented out The Darby in Midtown, New York for a crowd that included Adrien Brody, Kate Hudson, Bono and Jay-Z. Cabaret star Lady Rizo aka Amelia Zirin-Brown was the night’s emcee, who said, “Some of you know him as Gerry, other as Gerard. I know him as Mistress Butler,” according to Page Six.
Gerry sang the Oasis hit “Wonderwall” first, before asking John Mayer to join him onstage. John also ended up singing his hit “Who Says.”


text:
The Trouble With Critics
The trouble afflicting many critics of the Order of Nine Angles is two-fold. First, they seem unable to see “The Nexion” for “the nexions”. That is, they confuse a nexion – be it a sphere or spheres on the Tree of Wyrd (and the associated archetypes and Occult praxises of such spheres) or be it a group or lodge or temple of those associating themselves with the O9A – with The Nexion that is The Seven Fold Way and thus the O9A.
Second, they illogically generalize from the particular, assuming as they so often do that the opinion of someone associated with the ONA is “ONA policy”, thus ignoring as they do the fact that no one has the authority to speak or write “on behalf of the O9A” because there is no O9A “authority” and never was any O9A “authority”. There are only personal experiences, personal opinions, personal interpretations since the only real “authority” in the O9A is what the individual discovers, learns, via pathei mathos both esoteric and exoteric, when they undertake the anados that is the Seven Fold Way.
Similarly in respect of works about or inspired by the Order of Nine Angles. They have no “authority”. Which is why neither the Deofel Quartet nor any essay or essays about ‘the sinister feminine’ (or about anything else, such as the Seven Fold Way) represent O9A policy. They present only the the opinions, the interpretation(s), and perhaps the experiences, of their authors. And this applies even if the author is or was Mr Anton Long.
Given that all this has been explained many times in the past twenty and more years – including in the 1992 publication The Satanic Letters of Stephen Brown – then why do critics of the O9A continue to be so afflicted?
The answer is simple: because of their physis, their character. Some are so afflicted because they are territorial, needing to defend what they believe ‘satanism’ and/or the Left Hand Path are, with some of these types additionally claiming that the O9A is just “one man with a typewriter” and, latterly, “one man with a word-processor”, and now, recently, just “one man with a computer and an internet connection.”
Some are so afflicted because they have gotten lost in the O9A’s Labyrinthos Mythologicus and emerge confused, finding their bearings again by believing that the O9A is ‘fake’ or ‘not real’ or ‘a joke’.
Some are so afflicted because – to be blunt – they lack the intellect to grasp the difference between “The Nexion” and “the nexions” let alone grasp the subtle intricacies of the Seven Fold Way.
Some are so afflicted because they are just ignorant about the O9A, having prejudged it often on the basis of reading a few O9A texts and what others have written (usually on the internet) about the O9A.
Some are so afflicted because of hubris, sincerely believing that they “know all about the O9A” even though, when asked, they cannot answer questions from O9A Adepts regarding O9A esotericism. Lacking the honesty to admit that their knowledge about the O9A is limited, such critics either arrogantly dismiss such questions as “nonsense” or as “without meaning,” or ignore them. One of the markers enabling diagnosis of this type of physis is that they cannot read primary esoteric sources in their original language and have to resort to translations.
Some are so afflicted because they sincerely believe themselves to be very knowledgeable about the Occult and, believing they have discovered flaws in the O9A, trumpet their “findings” in order to establish a reputation for themselves and in the hope of attracting followers. When their “findings” or their knowledge are challenged, they invariably resort to argumentum ad nauseam and/or to argumentum ad hominem, with some of these types sincerely believing that they are on a “justified crusade” to “expose the ONA.” As with the ‘hubriatic type’, one of the markers enabling diagnosis of their physis is that they cannot read primary esoteric sources in their original language and have to resort to translations, which lack of direct knowledge of such primary sources reveals the limitations of their knowledge of matters Occult and O9A.
Finally, some are so afflicted because they have a personal agenda, such as a belief in the dogma of the Nazarene, or an irrational dislike of the person they fervently believe is behind the pseudonym “Anton Long”.
Of course, despite what we wrote at the beginning about the two-fold cause of such an affliction, many of the afflicted critics will continue to inflict their criticisms on others, given that for them their criticism of the O9A seems to have become a necessary part of their public (usually internet-created and internet-managed) persona.
And in the matter of the genesis and the spread of such an affliction, we were glad to help.

domain: literature


text:
Both beta-adrenergic blockade and bradycardia may contribute to the therapeutic effect of beta-blockers in chronic heart failure (CHF). This study tested the relative importance of bradycardia by comparing cilobradine (Cilo), a sinus node inhibitor, with a beta-blocker, metoprolol (Meto), in an established canine model of CHF. Dogs were chronically instrumented for hemodynamic and left ventricular (LV) volume measurements. CHF was created by daily coronary embolization via a chronically implanted coronary (left anterior descending coronary artery) catheter. After establishment of CHF, control (n = 6), Meto (30 mg/day, n = 5), Cilo (low) (1 mg/kg/day, n = 5), or Cilo (high) (3 mg/kg/day, n = 5) was given orally for 12 weeks. Systemic hemodynamics, echocardiography, and pressure volume analysis were measured at baseline, at CHF, and 3 months after treatment in an awake state. Protein levels of cardiac sarcoplasmic reticulum calcium-ATPase (SERCA2a), ryanodine receptor (RyR2), and Na(+)-Ca(2+) exchanger (NCX1) were measured by Western blot. RyR2 protein kinase A (PKA) phosphorylation was determined by back-phosphorylation. After 12 weeks, Meto and Cilo (high and low) produced similar bradycardic effects, accompanied by a significantly improved LV dP/dt versus control [Meto, 2602 +/- 70; Cilo (low), 2517 +/- 45; Cilo (high), 2579 +/- 78; control, 1922 +/- 115 mm Hg/s; p < 0.05]. Both Meto and Cilo (high) normalized protein levels of SERCA2a and NCX1 and reversed PKA hyperphosphorylation of RyR2, in contrast to controls. High-dose cilobradine effectively produced bradycardia and improved cardiac function after CHF, comparable with metoprolol. Restored protein levels of SERCA2a and improved function of RyR2 may be important mechanisms associated with cilobradine therapy.
Tags: analysis, Arteries, CHF, dogs, Echocardiography, heart, heart failure, hemodynamics, Metoprolol, model, phosphorylation, pressure, remodeling, Research, ryanodine, sarcoplasmic reticulum, therapy, Ventricular Function, volume, volume measurement

domain: medical


First Congress legislator switches to TRS after Telangana elections
news Monday, September 01, 2014 - 05:30
The News Minute| September 1, 2014| 04.45 pm IST The Congress in Telangana received a setback Sunday when its one of the legislators quit the party and announced his decision to join the ruling Telangana Rashtra Samithi (TRS). Koram Kanakaiah, a member of legislative assembly representing Yellandu in Khammam district, resigned from the main opposition party. He will be joining the TRS Monday. Kanakaiah, who was elected from Yellandu in the elections in held in April, had called on Telangana Chief Minister K. Chandrasekhar Rao two days ago. He is the first legislator to switch loyalties after the elections, which saw TRS storming to power to form first government in the newly-created Telangana state. Kanakaiah's resignation will bring down Congress' tally in 119-member assembly to 20. The TRS bagged 63 seats in the elections to form the government on its own. His resignation from the Congress came a day after the Telugu Desam Party (TDP) suffered a shock in the same district with the resignation of its senior leader T. Nageswara Rao. The former state minister quit TDP along with his followers and announced their decision to join the TRS. The resignations of TDP and Congress leaders and their joining of TRS is expected to strengthen the ruling party in Khammam district, where it is considered weak. In fact, TRS recorded its worst poll performance in Khammam district, bordering Andhra Pradesh. The party could win only one out of 10 assembly seats in the district. With IANS
Show us some love! Support our journalism by becoming a TNM Member - Click here.


text:
The Instant Vortex Plus Dual Drawer Air Fryer turns out crisp, crunchy treats such as fries and chicken wings every time. The two frying drawers ensure you can cook different foods at the same time, even syncing their finish times. It’s a versatile appliance, offering the ability to bake, roast, grill, reheat and dehydrate as well as air fry. It also comes with a window and light that allow you to keep an eye on food through cooking. This model is expensive, however, and while the overall capacity is large, each frying basket is small, reducing how much you can cook in the appliance.
Crisp, evenly browned results
Cook two foods at once
Windows let you check on food during cooking
Frying baskets hold a smaller quantity
Requires some experimentation
Why you can trust TechRadar We spend hours testing every product or service we review, so you can be sure you’re buying the best. Find out more about how we test.
Instant is a relative newcomer to the air fryer market, having launched its first model in 2019. The brand first appeared in 2010, unveiling the Instant Pot multicooker, and has quickly become one of the must-have brands in this space. Its top-of-the-range Instant Vortex Plus tops our list of the best air fryers you can buy.
Now it's overhauled its most expensive air fryer to offer a dual-zone version that lets you cook two different foods at once, while also ensuring cooking times can be synced so that both are ready at the same time.
Just like every other air fryer on the market, the Instant Vortex Plus Dual Drawer Air Fryer, which is known as the Instant Vortex Plus Dual Air Fryer in the US, circulates hot air around food to cook it, creating a crisp, crunchy finish. Instant claims food cooked in its air fryers requires 95% less oil than deep frying. Both drawers of the air fryer also feature a ClearCook window, which when coupled with the light inside of the cooking cavity, enable you to keep an eye on food without having to open the drawer and interrupt the cooking cycle.
The two drawers of the Instant Instant Vortex Plus Dual Drawer Air Fryer offer a 3.8-litre / 4-quart capacity, for 7.6 litres / 8 quarts in total, which Instant says is sufficient for eight portions. This makes this model the largest air fryer Instant offers. As well as air frying, this kitchen gadget can also roast, bake, grill, dehydrate and reheat food. It comes sporting a new angular design, with a smaller dial for adjusting the temperature and cooking duration.
Slightly larger than the Instant Vortex Plus, but not as bulky as its closest rival, the Ninja Foodi Max Dual Zone Air Fryer AF400UK, we found the Instant Vortex Plus Dual Drawer Air Fryer did an excellent job of evenly browning fries and chicken wings. However, with no cooking charts or guidance in the instructions, using it will require a degree of experimentation.
At £199.99 / $179.99, it’s the most expensive air fryer Instant offers; but for those who want to prepare two dishes at the same time, it’s worth the investment.
Instant Vortex Plus Dual Drawer Air Fryer price and availability
- List price: £199.99 / $179.99
The Instant Vortex Plus Dual Drawer Air Fryer, which is known as the Instant Vortex Plus Dual Air Fryer in the US, costs £199.99 / $179.99 and is available from Instant direct or through Amazon. This model sports a glossy all-black finish; however, it also comes in an option with stainless steel accents, which is priced at £219.99 / $199.99. This model isn’t currently available in Australia.
The air fryer's predecessor, the Instant Vortex Plus continues to be available worldwide, including Australia. It has a slightly smaller 5.7-litre / 6-quart capacity and is priced at £119.99 / $119.95 / AU$269.
- 7.6-litre / 8-quart capacity
- Angular design with touch controls
- Can switch between C or F for cooking temperature
The larger capacity of the Instant Vortex Plus Dual Drawer Air Fryer means it takes up slightly more countertop space than the Vortex Plus, measuring 31.7 x 40.3cm x 38.4cm /12.5 x 15.9 x 15.11 inches (h x w x d). However, as we’ve already mentioned, it isn’t as bulky as some other dual-zone air fryers on the market.
It comes with a new look, too. Its cuboid shape makes way for a more angular look, with the touch controls positioned closer to the top of the air fryer. The dial to adjust the cooking temperature and duration is slightly smaller here. Unfortunately, while at first glance the glossy black finish looks stylish and smart, it didn’t remain looking that way for long. It’s prone to picking up greasy fingerprints, and so will need to be regularly cleaned with a damp cloth.
As well as air fry, the Instant Vortex Plus Dual Drawer Air Fryer can roast, bake, grill, reheat and dehydrate. The air fryer has a total capacity of 7.6 litres / 8 quarts, which is split evenly across the two frying baskets. The front of the baskets feature a built-in plastic window, which, along with a light in the cooking cavity, ensures you can see how cooking is progressing. For easy cleaning, both the baskets and the crisper plates that sit inside them are dishwasher-safe – an improvement on its predecessor, where only the crisper plate could be cleaned in the dishwasher.
Cooking temperature ranges from 35 C / 95 F to 205 C / 400 F for up to 72 hours at a time – although the latter is at lower temperatures. At its maximum temperature, the Instant Vortex Plus Dual Drawer Air Fryer can cook for up to one hour. It’s also possible to adjust the cooking temperature in one-degree increments, compared to the three-degree increments offered by the Instant Vortex Plus. You can even switch between F and C for the cooking temperature.
When it comes to cooking duration, you can set each basket to complete cooking independently, or utilize the sync feature that stalls the basket with the shorter cooking time so that the two zones complete at the same time.
The air fryer doesn’t come with a recipe booklet. Instead, you’ll need to go online or use the Instant app for your smartphone or tablet for inspiration. There’s also no cooking chart or guides, which means some experimentation over the ideal cooking temperature and time will be required. This is especially true when cooking frozen foods, to ensure you can cook them to your liking.
- Crisp, evenly browned results
- Check on food with the help of window and light
- Requires some experimentation
The Instant Vortex Plus Dual Drawer Air Fryer has a bigger overall capacity than its predecessor, the Instant Vortex Plus, but each frying basket can hold only 4 quarts / 3.8 litres. Without any cooking charts or guides in the box, it was clear that we’d need to experiment with cooking temperatures and durations to ensure food was cooked perfectly.
We started with 450g / 1lb frozen steak cut fries in basket one, using the default air-frying settings: 196 C / 385 F for 15 minutes. Usefully, the air fryer automatically preheats for three minutes once a time and temperature has been selected, while also offering a visual and audible prompt to add food when it reaches temperature.
Once the cooking time had finished, however, it was clear the fries weren’t cooked to our liking – they were pale and soggy, with some still raw in the center. So we added a further three minutes to the cooking time. Frustratingly, the air fryer goes through the automatic preheat again, in effect pushing cooking time to an additional six minutes. However, the fries still weren’t crisp and crunchy enough, so we added a further two minutes (five with the preheat). The fries still weren’t properly cooked,, in spite of the additional time, so it was clear this wasn’t going to be a success, so we abandoned cooking.
For the next batch we raised the temperature to 204 C / 400 F (the same temperature for cooking chicken wings in the air fryer) and the duration to 15 minutes, but when the cooking time had come to an end, the fries still weren’t cooked through. We added another four (seven, including the preheat) but this still didn’t achieve the desired crunch and level of browning we were looking for.
It became apparent that the air fryer couldn’t cope with the usual quantity, so we reduced the batch to 300g / 10.5oz. This time the fries were spread across the frying basket in one layer, rather than being piled on top of each other as they were with the larger quantity. After 18 minutes, the results were evenly browned and crisp fries, with soft fluffy potato inside.
We fared better with chicken wings. Although 450g / 1lb of chicken wings was too much to arrange in one layer in the frying basket , they weren't as tightly packed as the fries, and the hot air was able to circulate better. They didn’t require any additional oil and following 21 minutes of cooking, we were presented with succulent wings with crisp, evenly browned skin.
When it came to homemade fries, we used our experience with the steak cut fries, placing only 300g / 10.5oz in the frying basket. We used 1.5tsp of oil, which is what the recipe called for. This time we set the air fryer to 191 C / 376 F for 20 minutes, the suggested cooking time for homemade fries in the Instant app. Once the cooking time had passed, the potato batons had been turned into crunchy, evenly browned fries that were soft and fluffy inside.
We also noted that when tipping out fries once they were cooked, the crisper plate didn’t fall out – something that regularly happened during testing of the Instant Vortex Plus.
Both frying baskets can be used at the same time. It’s possible to cook food at varying temperatures for a different duration in each drawer, while also ensuring that cooking completes at the same time, with the Sync Finish feature.
We were impressed by the ClearCook windows and light, which meant we could monitor cooking, checking just how brown our food was getting. Also useful was the reminder to shake function, which provides an audible and visual prompt to shake or turn the food so the hot air can reach every inch of it.
We didn’t get round to baking in the air fryer during testing, but we did note that instructions state that only metal or glass containers can be used with the bake setting. In addition, when placed in the air fryer there must be 2.5 cm /1 inch of space around said container. However, none of the our three most-used baking tins (an 8-inch circular tin for Victoria Sandwich cakes, a 1lb loaf tin and a 8-inch square brownie pan) would fit in either drawer of the Instant Vortex Plus Dual Drawer Air Fryer, let alone ensuring the required amount of space around them. As such, it’s unlikely we’d use the air fryer to bake.
Should I buy the Instant Vortex Plus Dual Drawer Air Fryer?
Buy it if...
You want to cook different foods at the same time
The two separate frying drawers of the Instant Vortex Plus Dual Drawer Air Fryer ensure you can cook two different foods at the same time, while the Sync Finish feature means they’ll be ready at the same time, too.
You want to keep an eye on food during cooking
The ClearCook windows let you check on the cooking progress of food, without having to open the frying drawer, which would affect the stability of the temperature in the cavity. The light makes it easier to view contents, too.
You want versatility
As mentioned, the Instant Vortex Plus Dual Drawer Air Fryer can do more than just air fry: it can roast, grill, bake, reheat and dehydrate as well. If you’re looking for one appliance that can do it all, rather than purchasing single-use kitchen gadgets, this is it.
Don't buy it if...
You’re on a budget
At $179.99 / £199.99, this is the most expensive air fryer Instant offers. If you’re on a tight budget then consider the Instant Vortex Plus, which has fewer cooking functions and only one frying drawer, but is considerably more affordable.
You cook large quantities
The Instant Vortex Plus Dual Drawer Air Fryer has an overall capacity of 7.6 litres / 8 quarts, but each drawer can hold a maximum of only 3.8 litres / 4 quarts. You’ll struggle to cook large quantities of different foods at the same time in this air fryer. If this is a requirement, consider the Ninja Foodi Max Dual Zone Air Fryer AF400UK instead, which has drawers that can hold 4.75 litres / 5 quarts each.
You want to use the air fryer to bake
While this air fryer does have a bake function, the smaller drawer size and the fact you must leave 2.5cm / 1 inch of space around baking tins means it won’t be the most effective air fryer for baking. Instead consider the Instant Vortex Plus, which has one larger drawer and can accommodate common-sized baking tins.
First reviewed: January 2022
- Check out more great air fryer deals
Carrie-Ann Skinner was formerly Homes Editor at TechRadar, and has more than two decades of experience in both online and print journalism, with 13 years of that spent covering all-things tech. Carrie specializes in smart home devices such as smart plugs and smart lights, as well as large and small appliances including vacuum cleaners, air fryers, stand mixers, and coffee machines. Carrie is now a copy editor at PWC.

domain: food


questt.com.cn,desktop laser engraver manufacturers,The minimum order quantity at QUESTT Industrial Laser Cleaning Machine Manufacturer is required, yet negotiable. To enable customers to get the products with high cost-performance ratio like desktop laser engraver manufacturers, we strongly recommend customers place a larger quantity of goods. The larger volume of orders customers place, the more favorable price they will get.
View More >>
QUESTT provides desktop laser engraver manufacturers products that are selling well in United States, Arabic,Turkey,Japan,German,Portuguese,polish,Korean,Spanish,India,French,Italian,Russian,etc.
QUESTT,Our company main produces fiber laser marking machine companies,fiber laser marking engraving machine,fiber laser marking machine in china.


text:
Excellent news. Maria Taylor’s first full collection Melanchrini is now available to order from Nine Arches Press. Editors Jane Commane and Matt Nunn recognised Maria’s talent a couple of years ago and offered her a publishing deal. Maria, who is also co-founder of Crystal Clear ( publishers of my pamphlet and producers of Hearing Voices magazine), a mother of twin four-year olds and a lecturer in creative writing, has worked hard to get this collection together. I first heard Maria read at the Word! open mike in Leicester, and was immediately struck by the quality and originality of her work. You can read some poems from the book on the Nine Arches website and once you do you will want to own a copy. Oh, and the cover is lovely too.
Melanchrini by Maria Taylor, available to order now


text:
"Wadhera and Wadhera's debut children's book uses Indian folklore to craft a fairy tale about believing in oneself and accomplishing the impossible.
India is threatened by an evil demon, Raakshasa, who wants to rule the entire country. The kings of the North and the South devise a plan with their royal adviser, Pundit, to defeat the demon by uniting their soon-to-be-born children against her. When Pundit explains it is the children's destiny to defeat Raakshasa, both kingdoms rejoice. After the heirs, Prince Amir and Princess Sonali, each turn 3, Pundit gives them magical rings that endow them with the ability to harness the power of Shakti, the supreme power of the universe, to use against the demon. Raakshasa, however, curses the princess's family as they leave the palace, turning them to stone. Sonali breaks the curse with her ring, and is then rescued by a cow and a peacock, who raise her in a nearby village. There, she thrives as an ordinary village girl, but believes that she's meant for greater things. Years later, when she encounters Amir, who possesses the secret to breaking the curse, they go on a mission to save her parents. Along the way, Amir falls in love with her bravery and intelligence. Sonali is presented as a brave, strong, and intelligent female character, who saves herself and her parents, with just a little help from Amir. She's a wonderful role model for young girls, and her independence and strength give a refreshing, modern twist to traditional fairy tales, in which the man usually saves the woman. The way that the prince and the princess unite to defeat a stronger foe will show young readers that they can accomplish the impossible if they believe and refuse to give up. The text is easy to follow, and the pictures are vibrant, expressive, and compelling, bringing the magic of the story to life.
A delightful foray into magic, Indian culture, and the power of belief."-Kirkus Reviews

domain: library


text:
Zig Zag Textile Platform Heels. Style: slip on peep toe platform shoes. NEXT . Size UK 5.5. New without box. gorgeous multi colour knitted textile pattern with cork heels & platforms. cork effect heels & platforms.
Next Signature Black Silver Bronze Textile Peep Toes High Slim Heeled Platform Shoes. Mainly vblack in colour with silver & bronze metallic textile upper. Silver coloured cushioned insole with Next Signature Logo.

domain: design


text:
Siskiyou Candidate Spotlight: Elizabeth Pyles
Updated: May 9
(MOUNT SHASTA, California ) Dave Theno, the program manager at Mount Shasta’s Golden Eagle Charter School, knows what it means to migrate north through California’s Great Central Valley, to settle on-up the Siskiyou trail. Alongside Theno, Golden Eagle’s Elizabeth Pyles migrated north from Chico over thirty years ago.
Their unique and tenured connection to the area equips them to meet the specific educational needs of Siskiyou County families. But has time actually turned them into locals?
“When you move somewhere like this, if you haven’t been here for generations, you’re still a newcomer,” Theno explains, smiling.
“Everyone here all knows each other from high school,” Pyles adds. “I moved here 33 years ago and I still feel like a transplant sometimes.”
Tucked away in the shadows of Mount Shasta, Siskiyou County communities harbor unique, built-in resources, but they also have specific needs. In order to best-serve the area, both Theno and Pyles understand how necessary it is to adapt in order to solve unique problems.
Golden Eagle’s history is rooted in the community’s desire for curriculum adapted specifically to the needs of the individual family. Living in Siskiyou County is sparse and isolated, with families seeking educational solutions aligned with their environment, values, and logistical complexities.
One of the educational challenges unique to places like Golden Eagle’s Mount Shasta location is helping students visualize and experiment with the possibilities of their future, regardless of whether that means staying or leaving. You have to be able to imagine life beyond a mountain you can’t see over or through.
“Things are pretty insular here, and you can grow up feeling constrained,” Theno explains. “The economy here doesn’t offer a ton of professional jobs. We have education, healthcare, law enforcement, and forest services, but there isn’t a rich tapestry of professional jobs, so you don’t grow up seeing a lot of modeling of great situations if you decide to stay.”
The malleability of Golden Eagle’s founding principles, however, allows Siskiyou County families to respond to developmental needs through variety and specificity.
“I’ve been here from the beginning,” Pyles adds. “We were always unique in that our parents could choose their curriculum, and the educational facilitators would pull out what the students were doing and put that into a learning record for the state. So that’s the concept that Golden Eagle has always tried to keep: parental choice and a unique curriculum experience. Most of the charter schools were going to one curriculum for all because it simplified things. But we tried to keep this variety and unique perspective to better serve the area.”
Pyles, an experienced classroom aide who has been with Golden Eagle since its inception, is doing exactly what Theno says the area needs: providing for Siskiyou county’s youth a model of unique professional development.
Pyles is pursuing an affordable job-embedded bachelor’s degree. She attends Reach University while also receiving coursework credit for her paid duties at Golden Eagle. Pyles has served Siskiyou County as both an educator and as a journalist, but the right path to a bachelor’s degree has eluded her for years.
She now pays $75 per month in tuition, and has been able to transform where she lives and works into her college campus.
Theno explains that the exploration of alternative career development has almost become a requirement for those wanting to live and work in the area. “Those of us who have been able to go into certain fields while also living here have definitely had to be crafty about how things work,” he says.
“I’ve definitely learned a lot about myself through this process,” Pyles says of her Reach experience. “I’m teaching and learning at the same time, and it’s taught me a lot about my own experiences as a student, and I think that has helped me become a more compassionate teacher.
“I’ve come to realize that I really want to work with kids one on one as a teacher. School can sometimes be a great source of anxiety for them, especially here, and I know what that feels like. I love helping them in that way, walking and talking with them. I haven’t figured everything out yet, though. It’s all still evolving.”
A willingness to adapt and evolve can serve as a consistent source of strength, and both Pyles and the community she serves have seen the benefits of creativity and perseverance. Efforts like hers showcase how rural and isolated areas can still produce their own talent pipelines. For Pyles, both Golden Eagle and Siskiyou County have been integral to her professional growth.
“I love Siskiyou County,” she says. “As a reporter, I’ve been to a lot of different places here talking to different people. A lot of history and diversity, all through Weed and Mount Shasta and Yreka. It’s just beautiful, and I absolutely love the people here.”
Candidate Elizabeth Pyles in her Golden Eagle classroom.

domain: geography


text:
- This event has passed.
2020 ADEA Diversity & Inclusion Workshop (Virtual)
October 28, 2020 @ 3:30 pm - 4:45 pm
You are invited to attend the virtual 2020 ADEA Diversity & Inclusion Workshop on Wednesday, Oct. 28 from 3:30 to 4:45 p.m. Eastern Time. New this year, the ADEA Diversity & Inclusion Workshop presents the first-ever ADEA Tapestry Table – a talk-show style panel with audience engagement and the goal of fostering dialogue and broadening perspectives on diversity, equity and inclusion in dental education.
Our inaugural ADEA Tapestry Table Workshop features guest Dominique Jackson, LGBTQIA+ activist and star of the FX network’s groundbreaking television series POSE, and highlights health and well-being issues within the LGBTQIA+ community with an emphasis on transgender individuals.
The ADEA Diversity & Inclusion Workshop is open to the public and free of charge.
Click here or below to register.

domain: sociology


text:
Brawn: Schumacher more than capable of winning
8 August 2012 – Michael Schumacher still has what it takes to win in F1, despite his poor run of form in 2012. That is the firm opinion of Mercedes Team Principal Ross Brawn, with the seven-time World Champion having faced much criticism since the Hungarian Grand Prix.
In Budapest, Schumacher parked his car one row further back on the grid than he should have, then switched off his engine through confusion at the consequential aborted start. He later retired for the sixth time this season, resulting in more condemnation for the German despite all of his problems bar one having been purely technical so far this year.
"So the fact is that something went wrong - and when something goes wrong it’s the responsibility of me and the team to not allow it to go wrong again.”
Mercedes are keen to retain Schumacher for a fourth season in 2013 and it is heavily suspected that the 43-year-old will make his decision during the current summer break.
“Very professional, very committed, good attitude, speed, work very well in the team, work well together as a pairing - which is not always easy with drivers - and I believe both are more than capable of winning races if we provide the equipment,” Brawn added of both Schumacher and team-mate Nico Rosberg. “That is all that you can ask from a driver.”
In Valencia for the European Grand Prix six weeks ago, Schumacher picked up his first podium finish since winning the Chinese Grand Prix for Ferrari in October 2006.

domain: sports


text:
It is impossible to ignore the significance of the fact that the Ford Motor Company, the one major car company that said “No Thanks” to Obama’s bailout of the auto industry, is now #1 – surpassing both Chrysler and GM, who did accept a “deal with the devil” and are still under the thumb of the Washington bureaucrats. Then again, Ford’s success should come as no surprise – the heavy hand of government has never resulted in the kind of innovation and drive that energizes free markets.
For example, after a BILLION dollars of R&D, including untold infusions of taxpayer money, GM has finally produced the Chevy Volt – an electric car that costs over $40,000 and goes only 40 miles on a charge, at which point it either has to be plugged into an outlet again, or rely on good old-fashioned petroleum energy to power it. Not exactly a “major breakthrough” in transportation history.
Note that these nitwits in the “environmental movement” always ignore the fact that the electricity that powers these vehicles comes from sources that they themselves hate – coal, oil, natural gas, and nuclear. Oh, I forgot – windmills and solar panels will magically replace all those “icky” fossil fuels. Right. But no matter, such a “green” car is naturally being touted by President Obama as the wave of the future.
And the reaction from car buyers? Despite all the hoopla from the Whitehouse, the Volt has produced so little demand that it is now offered with a $7,500 tax credit – presumably because only those making enough money to benefit from such a credit can even afford the car in the first place.
Meanwhile, over at Ford, they are going gang-busters. And while anger at GM and Chrysler for taking the Obama bailout has indeed resulted in many of their former customers now opting to buy a Ford, the real driving force (pun intended) behind Ford’s success is that they are ignoring “politically correct” but economically untenable boondoggles like electric cars. They are instead responding to the market by producing the kinds of vehicles that people actually want to buy.
Take Ford’s F-150 pickup truck, for decades it has been one of the most successful vehicles in automotive history. And when “green” zealots spouted their propaganda that such big, powerful trucks are “socially irresponsible,” Ford responded by equipping the F-150 with even bigger, more powerful models, like the supercharged Lightning and the soon to be released 500 horsepower Raptor. Even in naming their vehicles Ford thumbs their nose at the P/C crowd.
Speaking of all-time great names for a car, there is the Ford Mustang – the 2011 model now sports an all new, super high-tech 5.0 liter V-8 motor that cranks out 412 rip-roaring horsepower, does 0-60 in less than 5 seconds, and basically shouts to the rest of the world: “Eat my dust!”
I drove one the other day, a 2011 Mustang convertible with the 6-speed manual transmission, and after a lifetime of owning high-performance cars from ’60’s muscle cars to a Turbo Porsche, I can tell you, this is one amazing vehicle. Docile and smooth around town, you have no idea what is lurking under the hood…until you stomp on the gas. Then all hell breaks loose (as do the rear tires). The V-8 howls, 7000 rpm comes up in the blink of an eye, and you are at triple-digit speeds in only a few seconds more. And for those who view 400 horsepower as merely “adequate” – the Mustang GT-500 model comes with a 5.4 liter, Supercharged motor pumping out a whopping 550 horsepower.
In other words, the Mustang is a real car-lover’s car – one that is actually FUN to drive. Not some tepid, mundane, and ultimately boring piece of environmentalist nonsense. Ask yourself, how many real, red-blooded Americans would actually want to drive a Prius? After all, it is probably no coincidence that there are a rather disproportionate number of Priuses, Honda Insights, and Smart Cars adorned with “Obama ’08” bumper stickers.
Even Ford’s minor concessions to the “green” movement are done with a wink and a nod to their customers. The Ford Taurus, a big, comfortable luxury sedan, can be had with an Eco-Boost motor. Now, while the “greenies” will swoon over the “Eco” part of the logo, those in the know understand that it is that deliciously enticing second word that tells the tale – “boost” in this case means twin-turbochargers making 365 horsepower (soon to be upped to an even more politically incorrect 415 horsepower). And it produces performance that is nothing short of astonishing in such a large and relatively heavy car.
And after buyers told Ford they wanted a bit more power in the Fusion, a mid-size sedan that competes with the Chevy Malibu, Toyota Camry, and Honda Accord, Ford responded by announcing that a version of the Taurus “Eco-Boost” motor will now be available in the new Fusion GT. It will pump out 340 horsepower, likely making it one of the fastest mid-size sedans on the market, yet at a price far below the likes of a BMW or an Audi. Oh, sure, Ford cleverly offers “Hybrid” version of the Fusion – it got Motor Trend Car of the Year for 2010 – but they know that the majority of buyers will choose power and performance over political correctness every time.
You see, Ford’s success is not that difficult to explain – unlike Obama and the liberal Democrats in Congress, Ford actually listens to its customers and gives them what they want, and the American people show their approval by voting with their dollars. Meanwhile, as they continue to ignore the will of the American people, the approvals of Obama and the Democrat dominated Congress continue to plummet.
There’s a lesson in all this, Mr. President…if you and your band of mirthless “progressives” care to listen.

domain: politics