text:
Australia's current reserve system represents a significant conservation achievement, consisting of more than 10,000 reserves covering over 16% of the country. However, it needs to be expanded further to ensure the survival of many native species. Currently, only 30% of threatened species have an adequate amount of their habitat protected in reserves. However, scientific estimates indicate that, by building on the current reserve system, we could provide adequate habitat protection for all threatened species by placing as little as 18% of the continent in strategically chosen reserves.
The reserve system also needs further development to ensure that it protects Australia's diverse landscapes. The goal of the National Reserve System is to develop and manage a comprehensive, adequate and representative national system of protected areas that contains samples of the full range of regional ecosystems in Australia. These areas need to be sufficiently large and well-designed to ensure that ecological communities, populations and ecological processes are maintained in the long term, and are resilient to the threats they face. In order to meet this goal, more work is required to fill gaps in the representation of significant ecosystems.
Australia is divided into 88 bioregions, which are large areas that are characterised by particular natural features, geology and environmental processes, and distinctive ecosystems and species. These are further divided into subregions. Based on 2012 figures from the Collaborative Australian Protected Area Database (CAPAD), all 88 Australian bioregions have some representation within the reserve system, but 34 of these currently have less than 10% of their area protected. At a finer scale, more than half of Australia's 419 subregions have less than 10% of their area protected, with 37 of these having no protection at all.
Maps showing the level of protection of bioregions across Australia, and further data about Australia's protected areas, are available from the Federal Government's National Reserve System website.
NPAC believes that Australians need a national park acquisitions program that can:
Deliver core habitat protection and connectivity to other protected areas
Leverage funds from State Governments, Conservancies and Indigenous conservation partners committed to enduring conservation management
Harness the commitment and passion of Traditional Owners for looking after their country and deliver cross-portfolio Indigenous outcomes
NPAC's 'Jewel in the Crown' campaign provides a vision of a national park legacy for all Australians, and includes a number of recommendations for reinvigorating and prioritising the national parks system.


text:
Q: How can I actually use delimeter? How can I actually use delimeter in java, this is the example data.dat file
   1 yuri
   2 fisher
   3 McMillan
   4 price
   5 soap

how can I only get the first numbers in every lines in the file and using useDelimiter() method
how can I do that to this code
Scanner src = new Scanner(fin);
                                 src.useDelimiter(" \n");
                                DefaultListModel model = new DefaultListModel();
                                while (src.hasNext()) {
                                String lol = src.nextLine();
                                model.addElement(lol);
                                }
                                list.setModel(model);
                                }
                                });

This is the whole code.. Uhmm.. It's not always 'lol' in the file I'll change It later
import java.awt.event.ActionEvent;
import java.awt.event.ActionListener;
import java.io.FileNotFoundException;
import java.io.FileReader;
import java.util.Scanner;
import java.util.logging.Level;
import java.util.logging.Logger;
import javax.swing.DefaultListModel;
import javax.swing.DefaultListSelectionModel;
import javax.swing.ListSelectionModel;
import javax.swing.event.ListSelectionEvent;
import javax.swing.event.ListSelectionListener;

/*
 * To change this template, choose Tools | Templates
 * and open the template in the editor.
 */

/**
 *
 * @author Jfetizanan
 */
@SuppressWarnings("serial")
public class IOtestForm extends javax.swing.JFrame {

    /**
     * Creates new form IOtest
     */
    public IOtestForm() {
        initComponents();
    }

    /**
     * This method is called from within the constructor to initialize the form.
     * WARNING: Do NOT modify this code. The content of this method is always
     * regenerated by the Form Editor.
     */
    @SuppressWarnings("unchecked")
    // <editor-fold defaultstate="collapsed" desc="Generated Code">
    private void initComponents() {

        jScrollPane1 = new javax.swing.JScrollPane();
        list = new javax.swing.JList();
        input0 = new javax.swing.JTextField();
        load = new javax.swing.JButton();

        setDefaultCloseOperation(javax.swing.WindowConstants.EXIT_ON_CLOSE);

        jScrollPane1.setViewportView(list);
        DefaultListSelectionModel m = new DefaultListSelectionModel();
        m.setSelectionMode(ListSelectionModel.SINGLE_SELECTION);
        m.setLeadAnchorNotificationEnabled(false);
        list.setSelectionModel(m);
        list.addListSelectionListener(new ListSelectionListener() {
            @Override
        public void valueChanged(ListSelectionEvent e) {
        int selected[] = list.getSelectedIndices();
        for (int i = 0; i < selected.length; i++) {
        String element = (String) list.getModel().getElementAt(selected[i]);
        input0.setText(element);
        }}});

        load.setText("Load");
        load.addActionListener(new ActionListener() {
                            @Override
            @SuppressWarnings("unchecked")
                            public void actionPerformed(ActionEvent e) {
                                 FileReader fin = null;
                            try {
                                 fin = new FileReader("data.dat");
                                } catch (FileNotFoundException ex) {
                                   Logger.getLogger(IOtestForm.class.getName()).log(Level.SEVERE, null, ex);
                                }
                                Scanner src = new Scanner(fin);
                                src.useDelimiter("\\s*lol\\s*");
                                DefaultListModel model = new DefaultListModel();
                                while (src.hasNext()) {
                                String lol = src.next();
                                model.addElement(lol);
                                }
                                list.setModel(model);
                                }
                                });

        javax.swing.GroupLayout layout = new javax.swing.GroupLayout(getContentPane());
        getContentPane().setLayout(layout);
        layout.setHorizontalGroup(
            layout.createParallelGroup(javax.swing.GroupLayout.Alignment.LEADING)
            .addGroup(layout.createSequentialGroup()
                .addContainerGap()
                .addGroup(layout.createParallelGroup(javax.swing.GroupLayout.Alignment.LEADING)
                    .addComponent(jScrollPane1, javax.swing.GroupLayout.DEFAULT_SIZE, 380, Short.MAX_VALUE)
                    .addComponent(input0)
                    .addComponent(load, javax.swing.GroupLayout.Alignment.TRAILING, javax.swing.GroupLayout.DEFAULT_SIZE, javax.swing.GroupLayout.DEFAULT_SIZE, Short.MAX_VALUE))
                .addContainerGap())
        );
        layout.setVerticalGroup(
            layout.createParallelGroup(javax.swing.GroupLayout.Alignment.LEADING)
            .addGroup(layout.createSequentialGroup()
                .addContainerGap()
                .addComponent(jScrollPane1, javax.swing.GroupLayout.PREFERRED_SIZE, javax.swing.GroupLayout.DEFAULT_SIZE, javax.swing.GroupLayout.PREFERRED_SIZE)
                .addGap(13, 13, 13)
                .addComponent(load)
                .addPreferredGap(javax.swing.LayoutStyle.ComponentPlacement.UNRELATED)
                .addComponent(input0, javax.swing.GroupLayout.PREFERRED_SIZE, javax.swing.GroupLayout.DEFAULT_SIZE, javax.swing.GroupLayout.PREFERRED_SIZE)
                .addContainerGap(94, Short.MAX_VALUE))
        );

        pack();
    }// </editor-fold>

    /**
     * @param args the command line arguments
     */
    public static void main(String args[]) {

        try {
            for (javax.swing.UIManager.LookAndFeelInfo info : javax.swing.UIManager.getInstalledLookAndFeels()) {
                if ("Nimbus".equals(info.getName())) {
                    javax.swing.UIManager.setLookAndFeel(info.getClassName());
                    break;
                }
            }
        } catch (ClassNotFoundException | InstantiationException | IllegalAccessException | javax.swing.UnsupportedLookAndFeelException ex) {
            java.util.logging.Logger.getLogger(IOtestForm.class.getName()).log(java.util.logging.Level.SEVERE, null, ex);
        }

        java.awt.EventQueue.invokeLater(new Runnable() {

            @Override
            public void run() {
                new IOtestForm().setVisible(true);
            }
        });
    }
    // Variables declaration - do not modify
    private javax.swing.JTextField input0;
    private javax.swing.JScrollPane jScrollPane1;
    private javax.swing.JList list;
    private javax.swing.JButton load;
    // End of variables declaration
}

A: You can use src.useDelimiter("\\s*lol\\s*"). See http://ideone.com/kZUL6 for an example of parsing.

A: An different solution would be splitting each the line at whitespaces:
String[] split = lol.trim().split("\\s+");

Then you should see the first numbers at each split[0].

A: Let's start with the basic
To load the file, you would do this
Scanner src = new Scanner(is);
while (src.hasNext()) {

    int line = src.nextInt();
    String value = src.next();

    System.out.println(line + " = " + value);

}

Now, what we really need is some way to model this information.  A better solution would be to create a Object of some kind that represents this information that makes it easier to manage.
Something like
public class LineOfText {

    private int line;
    private String value;

    public LineOfText(int line, String value) {
        this.line = line;
        this.value = value;
    }

    public int getLine() {
        return line;
    }

    public String getValue() {
        return value;
    }

}

Okay, then we need to apply the properties from the file to it.
Scanner src = new Scanner(is);
while (src.hasNext()) {

    int line = src.nextInt();
    String value = src.next();

    LineOfText lot = new LineOfText(line, value);

}

This we can add to the ListModel you've already created.
model.addElement(lot);

Now if we display this on our UI, it doesn't look very pretty.  What we really need is some way to "render" the text more efficiently.
Something like
public class LineOfTextListCellRenderer extends DefaultListCellRenderer {

    @Override
    public Component getListCellRendererComponent(JList list, Object value, int index, boolean isSelected, boolean cellHasFocus) {

        if (value instanceof LineOfText) {

            value = ((LineOfText)value).getLine();

        }

        return super.getListCellRendererComponent(list, value, index, isSelected, cellHasFocus);

    }

}

Should do.  We need to apply this renderer to the list you created earlier.
list.setListCellRenderer(new LineOfTextListCellRenderer());

Now, if we display this on the UI, we should only see the line numbers in the list.
Next, we need some way to detect the change of selection. For this we need a ListSelectionListener
list.addListSelectionListener(new ListSelectionListener() {

    @Override
    public void valueChanged(ListSelectionEvent e) {

        Object item = list.getSelectedItem();
        if (item != null && item instance of LineOfText) {
            ListOfText lot = (LineOfText)lot;
            // Now we have access to the text value
            // Simply use "setText" on the text area to apply it

            textArea.setText(lot.getValue());

            // Remember, "textArea" should be replaced with the name
            // of your text area
        }

    }

});

Now you see why it was a separate question ;)

A: set the delimiter to \\s*lol\\s*
public static void main(String[] args)
{
    String myString = "1 lol\n2 lol\n3 lol\n4 lol\n5 lol";
    System.out.println(myString);
    Scanner scanner = new Scanner(myString);
    scanner = scanner.useDelimiter("\\s*lol\\s*");
    while(scanner.hasNext())
    {
        System.out.println(scanner.next());
    }
}

redpajama_set_name: RedPajamaStackExchange


text:
Research ArticleCardiology Free access | 10.1172/JCI35470
ABCG1 and HDL protect against endothelial dysfunction in mice fed a high-cholesterol diet
Naoki Terasaka,1 Shuiqing Yu,2 Laurent Yvan-Charvet,1 Nan Wang,1 Nino Mzhavia,2 Read Langlois,1 Tamara Pagler,1 Rong Li,1 Carrie L. Welch,1 Ira J. Goldberg,2,3 and Alan R. Tall1
1Division of Molecular Medicine, 2Division of Cardiology, and 3Division of Preventive Medicine and Nutrition, Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York, USA.
Address correspondence to: Naoki Terasaka, Division of Molecular Medicine, Department of Medicine, Columbia University, PS 8-401, West 168th St., New York, New York 10032, USA. Phone: (212) 305-5789; Fax: (201) 305-5052; E-mail: nt2188@columbia.edu.
Find articles by Terasaka, N. in: JCI | PubMed | Google Scholar
Find articles by Yu, S. in: JCI | PubMed | Google Scholar
Find articles by Yvan-Charvet, L. in: JCI | PubMed | Google Scholar
Find articles by Wang, N. in: JCI | PubMed | Google Scholar
Find articles by Mzhavia, N. in: JCI | PubMed | Google Scholar
Find articles by Langlois, R. in: JCI | PubMed | Google Scholar
Find articles by Pagler, T. in: JCI | PubMed | Google Scholar
Find articles by Li, R. in: JCI | PubMed | Google Scholar
Find articles by Welch, C. in: JCI | PubMed | Google Scholar
Find articles by Goldberg, I. in: JCI | PubMed | Google Scholar
Find articles by Tall, A. in: JCI | PubMed | Google Scholar
First published October 16, 2008 - More info
First published October 16, 2008 - Version history
Received: February 27, 2008; Accepted: September 10, 2008
Plasma HDL levels are inversely related to the incidence of atherosclerotic disease. Some of the atheroprotective effects of HDL are likely mediated via preservation of EC function. Whether the beneficial effects of HDL on ECs depend on its involvement in cholesterol efflux via the ATP-binding cassette transporters ABCA1 and ABCG1, which promote efflux of cholesterol and oxysterols from macrophages, has not been investigated. To address this, we assessed endothelial function in Abca1–/–, Abcg1–/–, and Abca1–/–Abcg1–/– mice fed either a high-cholesterol diet (HCD) or a Western diet (WTD). Non-atherosclerotic arteries from WTD-fed Abcg1–/– and Abca1–/–Abcg1–/– mice exhibited a marked decrease in endothelium-dependent vasorelaxation, while Abca1–/– mice had a milder defect. In addition, eNOS activity was reduced in aortic homogenates generated from Abcg1–/– mice fed either a HCD or a WTD, and this correlated with decreased levels of the active dimeric form of eNOS. More detailed analysis indicated that ABCG1 was expressed primarily in ECs, and that these cells accumulated the oxysterol 7-ketocholesterol (7-KC) when Abcg1–/– mice were fed a WTD. Consistent with these data, ABCG1 had a major role in promoting efflux of cholesterol and 7-KC in cultured human aortic ECs (HAECs). Furthermore, HDL treatment of HAECs prevented 7-KC–induced ROS production and active eNOS dimer disruption in an ABCG1-dependent manner. Our data suggest that ABCG1 and HDL maintain EC function in HCD-fed mice by promoting efflux of cholesterol and 7-oxysterols and preserving active eNOS dimer levels.
Endothelial dysfunction is a key feature of early atherosclerotic lesions in both humans and animal models (1–3). It is characterized by decreased eNOS activity and NO bioavailability and increased expression of cell adhesion molecules such as VCAM-1 and ICAM-1, promoting atherosclerotic lesion formation, impaired blood flow, and thrombus formation. In animal models, increased dietary cholesterol plays a central role in inducing endothelial dysfunction (4–6). Dietary oxysterols, particularly 7-oxysterols, appear to have a key role in inducing decreased NO-induced vascular relaxation (7, 8). 7-Ketocholesterol (7-KC) is detected at high levels in human atherosclerotic plaques and in the plasma of patients with a high cardiovascular risk, and is abundant in oxidized LDL (9–11). In addition, oxysterols may be present in the diet and incorporated into plasma lipoproteins (12, 13). Dietary sources of oxysterols are cholesterol-rich foods (dairy, egg, meat products), especially those products that are heated in air during processing or are stored for long periods (14, 15). Thus, many foods in the Western diet (WTD) contain cholesterol oxidation products.
Plasma HDL levels are inversely related to the incidence of athero-thrombotic disease (16, 17). A part of the atheroprotective effect of HDL may be related to its role in preserving endothelial function (18, 19). The beneficial effects of HDL on ECs may include stimulation of proliferation, cell survival, migration, and NO synthesis as well as inhibition of the expression of VCAM-1 and ICAM-1 (20–23). HDL may have a specific role in reversing decreased eNOS activity in human ECs treated with oxidized LDL (24) or in reversing the decrease in eNOS-dependent vascular relaxation induced by high-cholesterol diets (HCDs) (4). The ability of HDL to cause relaxation of vascular rings has been reported to be impaired in scavenger receptor B-I–deficient (SR-BI–deficient) mice, and SR-BI expression in cultured cells enables an increase in eNOS activity in response to HDL through a mechanism that depends on the cholesterol efflux properties of HDL (25). While ATP-binding cassette transporters ABCA1 and ABCG1 have a major role in inducing cellular cholesterol efflux (26–28) and are known to be expressed in ECs (29), to our knowledge their role in preserving endothelial function has not been explored. ABCA1 mediates cholesterol efflux to lipid-poor apoA-I but only modestly increases cholesterol efflux to HDL (28, 30, 31). In contrast, ABCG1 promotes macrophage cholesterol efflux to HDL but not to lipid-poor apoA-I (28, 32–34). ABCG1 was recently shown to have a specific role not shared by ABCA1 in promoting efflux of 7-oxysterols from macrophages and transfected cells to HDL (28, 35). To better understand the adverse effects of dietary cholesterol and 7-oxysterols on endothelial function (7, 8), the present study was undertaken to test the hypothesis that ABCG1 and/or ABCA1, by promoting efflux of sterols and oxysterols from ECs, plays a key role in preserving eNOS activity in animals fed HCDs. Our studies show a major role for ABCG1 in defending endothelial eNOS activity in mice fed HCDs regulated to the efflux of cholesterol and 7-oxysterols and the preservation of eNOS dimer.
Impact of ABC transporter deficiency on endothelium-dependent vasorelaxation. Abcg1–/– and control mice were placed on a HCD (1.25% cholesterol, 7.5% cocoa butter, and 0.5% sodium cholate). After 11 weeks, both groups developed a similar moderate hypercholesterolemia (control, 331 ± 34 mg/dl; Abcg1–/–, 321 ± 46 mg/dl). To test vascular function in these mice, femoral arteries were preconstricted with phenylephrine, and relaxant responses to endothelium-dependent ACh and endothelium-independent sodium nitroprusside (SNP) vasodilating agents were measured. Arterial vasorelaxation in response to ACh was markedly attenuated in Abcg1–/– mice (Figure 1A). The ACh dose-response curve was shifted to the right in Abcg1–/– mice, and the maximum relaxation response was significantly reduced compared with arteries from control mice (P < 0.01; Figure 1B). In contrast, there was no significant difference in relaxation in response to SNP (Figure 1C). There was also no significant difference in ACh-induced or SNP-induced arterial relaxation in WT and Abcg1–/– mice fed the chow diet (Figure 1, D and E). We also assessed ACh-induced vascular relaxation in WTD-fed mice with single or combined deficiencies of ABCA1 and ABCG1 (Figure 1, F and G). This revealed a similar severe defect in vascular relaxation in Abcg1–/– and Abca1–/–Abcg1–/– mice (EC50, 79.6 ± 13.0 vs. 88.3 ± 14.7 nM), while the response of Abca1–/– mice (EC50, 42.1 ± 11.8 nM) was intermediate between these groups and that of the controls (17.4 ± 2.7 nM) (Figure 1F). There was no difference in relaxation in response to SNP in any of the groups (Figure 1G). These findings suggest that while both transporters may be involved in preserving vascular relaxation responses, ABCG1 has a more prominent role than ABCA1.
Response to vasoconstrictive agents in the femoral arteries from control (WT), Abcg1–/–, Abca1–/–, and Abca1–/–Abcg1–/– mice. (A–C) WT and Abcg1–/– mice (n = 4 per group) were put on a HCD (1.25% cholesterol, 7.5% cocoa butter and 0.5% sodium cholate) for 11 weeks. (A) Original trace recordings showing vessel tension increase after addition of 3 μM phenylephrine (PE) and relaxation in response to different concentrations of ACh. ACh-induced vasorelaxation occurred at the indicated concentrations in control and Abcg1–/– mice. (B) Vasorelaxation in response to ACh was markedly attenuated in Abcg1–/– mice. (C) There was no significant difference in relaxation in response to SNP. (D and E) WT and Abcg1–/– mice (n = 5 per group) were put on a chow diet. There was no difference between the groups in the response to ACh (D) or SNP (E). (F and G) WT, Abcg1–/–, Abca1–/–, and Abca1–/–Abcg1–/– (n = 5 per group) were put on a WTD (0.25% cholesterol and 21% milk fat) for 12 weeks. (F) There was a similar severe defect in vascular relaxation response to ACh in Abcg1–/– and Abca1–/–Abcg1–/– mice, while the response of Abca1–/– mice was intermediate between these groups and the controls. (G) There was no significant difference in relaxation in response to SNP. The results are represented as mean ± SEM.
Table 1 summarizes the effect of diet on vascular relaxation parameters in WT and Abcg1–/– mice. In the control group, the response to ACh was similar on the chow or WTD (Table 1) but impaired in response to the HCD (Table 1). There was a progressive, more marked impairment with increasing dietary cholesterol content in the Abcg1–/– mice (Table 1). The EC50 value for the response to ACh was approximately 4-fold greater than control in Abcg1–/– mice on the WTD, and 10-fold greater on the HCD, while there was no difference in response to the chow diet (Table 1). The data indicate that ABCG1 plays a progressively more important role in maintaining endothelium-dependent vasorelaxation as dietary cholesterol content is increased.
EC50 values of vasorelaxation induced by ACh in femoral arteries from control and Abcg1–/– mice a chow diet, WTD, or HCD
Cholesterol and 7-KC accumulation in aorta: effects of diet and genotype. In view of the specific role of ABCG1 in efflux of 7-oxysterols from cells (35), we next measured the content of cholesterol and 7-KC in non-atherosclerotic thoracic and abdominal aortas excluding the proximal aorta. Total cholesterol content was increased by the HCDs in a dietary cholesterol concentration–dependent manner (Figure 2A). However, there was no significant difference in cholesterol content between the control and Abcg1–/– mice (Figure 2A). 7-KC was not detectable in aortas of chow-fed mice but accumulated in response to the HCD (Figure 2B) and WTD (Figure 2, B and E). Accumulation of 7-KC was more prominent in Abcg1–/– mice (Figure 2, B and E). The ratio of 7-KC to cholesterol was significantly higher in Abcg1–/– mice than in controls in response to HCD (Figure 2C) and WTD feeding (Figure 2F). We also compared aortic cholesterol and 7-KC contents in Abcg1–/– mice with those in Abca1–/– and Abca1–/–Abcg1–/– mice in response to the WTD. There was no significant difference in cholesterol content between the control and Abcg1–/– or Abca1–/– mice (Figure 2D), while in Abca1–/–Abcg1–/– mice cholesterol content was significantly higher than in the controls (Figure 2D). In contrast, 7-KC was significantly increased in Abcg1–/– and Abca1–/–Abcg1–/– mice compared with controls, although no difference was found between Abcg1–/– and Abca1–/–Abcg1–/– (Figure 2E). The ratio of 7-KC to cholesterol was also significantly increased in Abcg1–/– and Abca1–/–Abcg1–/– mice (Figure 2F). Thus, we conclude that deficiency of both ABCA1 and ABCG1 results in increased cholesterol accumulation compared with accumulation associated with a single deficiency of the transporters, while accumulation of 7-KC specifically reflects deficiency of ABCG1. The latter finding parallels the impairment of vasodilatory responses and suggests that the impaired ACh-induced vascular relaxation in Abcg1–/– mice could be brought about by aortic accumulation of 7-oxysterols.
Cholesterol and 7-KC contents in thoracic and abdominal aortas: effects of diet and genotype. (A–C) WT and Abcg1–/– mice were put on a chow diet, WTD, or HCD (n = 4–5 per group). (D–F) WT, Abca1–/–, Abcg1–/–, and Abca1–/–Abcg1–/– mice were put on the WTD (n = 5 per group). (A and D) Cholesterol (Chol), (B and E) 7-KC, and (C and F) the ratio of 7-KC to cholesterol were measured. The results are represented as mean ± SEM. *P < 0.05 versus chow diet; †P < 0.05 versus WTD.
eNOS protein expression and dimerization in aorta. Previous studies have shown that the formation of eNOS homodimers is necessary for eNOS activity (36, 37). In response to the HCD, eNOS dimer levels were dramatically reduced in Abcg1–/– mice (Figure 3, A and B). Total eNOS and phospho-eNOS levels were also moderately decreased in Abcg1–/– mice (Figure 3, C and D), but the ratio of phospho-eNOS to eNOS did not change. On the WTD, Abca1–/–, Abcg1–/–, and Abca1–/–Abcg1–/– mice exhibited decreased eNOS dimer levels in aortas (Figure 3, E and F). This reduction was most prominent in Abcg1–/– and Abca1–/–Abcg1–/– mice (Figure 3, E and F). There was no difference in eNOS or phospho-eNOS levels between the groups (Figure 3, G and H). In aortas of chow-fed mice, there was no significant difference in eNOS dimer levels, eNOS, or phospho-eNOS levels between the control and Abcg1–/– mice (Figure 3, I–L). PECAM levels were not changed in any groups or diets (Figure 3, C, G, and K), indicating an intact endothelium. These data suggest that endothelial dysfunction induced by ABCG1 deficiency in response to the HCD resulted from the reduction of eNOS dimer levels.
Western blot for eNOS protein of mouse aorta. (A–D) Aortas from HCD-fed WT and Abcg1–/– mice. (E–H) Aortas from WTD-fed WT, Abca1–/–, Abcg1–/–, and Abca1–/–Abcg1–/– mice. (I–L) Aortas from chow-fed WT and Abcg1–/– mice. (A, E, and I) Western blot for eNOS dimer levels. (B, F, and J) Quantification of eNOS dimer/monomer levels. (C, G, and K) Western blot for eNOS and phospho-eNOS. (D, H, and L) Quantification of eNOS (filled bars) and phospho-eNOS (open bars). The results are represented as mean ± SEM. *P < 0.05 versus control.
ABCG1 expression and accumulation of 7-KC in aorta. To further evaluate the role of ABCG1 in endothelium-dependent vasorelaxation, we investigated ABCG1 expression in non-atherosclerotic aorta of Abcg1–/– mice that harbor a lacZ cassette insertion at the Abcg1 locus. Blue nuclear lacZ expression was detected specifically in ECs (Figure 4A, arrowheads), but not in other cells indicated by nuclear fast red staining (Figure 4A, arrows). We also carried out PECAM staining in aorta of WTD-fed Abcg1–/– mice, which indicated an intact endothelium (Figure 4B). As expected, these segments of abdominal and thoracic aorta did not show any evidence of atherosclerosis or macrophage accumulation (data not shown). We also measured NOS activity using aortic lysates in WTD-fed WT and Abcg1–/– mice. The NOS activity in Abcg1–/– mice was significantly decreased (Figure 4C). These data are consistent with the reduction of eNOS dimer levels in aorta (Figure 3, E and F). We also isolated ECs from aorta in WTD-fed WT and Abcg1–/– mice using an affinity column with anti-PECAM antibody. After isolation of ECs, PECAM, eNOS, and Abcg1 mRNA levels were increased by 15- to 20-fold compared with the non-endothelial fraction (data not shown). There was no significant difference in cholesterol content between WT and Abcg1–/– mice (Figure 4D). 7-KC levels (Figure 4E) and the 7-KC/cholesterol ratio (Figure 4F) were significantly increased in the ECs isolated from Abcg1–/– mice, but not in the non-EC fraction. These findings suggest that lack of ABCG1 in ECs leads to 7-KC accumulation and reduced eNOS dimer levels and that decreased eNOS activity is responsible for impaired vascular relaxation in mice fed HCDs.
LacZ expression in Abcg1–/– mice and NOS activity and sterol mass in WTD-fed WT and Abcg1–/– mice. (A) LacZ expression in endothelium of aorta in Abcg1–/– mouse. Blue nuclear lacZ expression was detected specifically in ECs (arrowheads) but not in other cells indicated by nuclear fast red (arrows). (B–F) WT and Abcg1–/– mice were put on a WTD for 12 weeks (n = 4 per group). (B) PECAM-immunostained aorta. Original magnification, ×200 (A), ×100 (B). (C) Aortic NOS activity. (D) Cholesterol mass, (E) 7-KC mass, and (F) 7-KC/cholesterol ratio in ECs and non-ECs from mouse aortas. The results are represented as mean ± SEM.
Effects of HDL and 7-KC on eNOS dimer and NOS activity. To further investigate the role of HDL and ABCG1 in promoting efflux of 7-oxysterols and preserving eNOS dimer levels and activity, we carried out experiments using human aortic ECs (HAECs), which are known to have a high level of ABCG1 (29). We first tested the effects of different concentrations of 7-KC (5–40 μg/ml) and HDL (100 μg/ml). 7-KC (5–40 μg/ml) significantly reduced eNOS dimer levels (Figure 5, A and B). Treatment of cells with HDL (100 μg/ml) following exposure to 7-KC prevented disruption of eNOS dimer levels by 7-KC (Figure 5, A and B). Only treatment with a high concentration of 7-KC (40 μg/ml) reduced eNOS and phospho-eNOS levels (Figure 5, A and C), and this did not change the ratio of phospho-eNOS to eNOS. The 7-KC concentration of 40 μg/ml also reduced eNOS mRNA (Supplemental Figure 1A; supplemental material available online with this article; doi: 10.1172/JCI35470DS1) and induced apoptosis (Supplemental Figure 1B), but this was not observed at lower concentrations. Notably the concentration range of 5–10 μg/ml led to 7-KC levels that were comparable with those in isolated ECs from Abcg1–/– mice (see below). HDL treatment completely preserved eNOS dimer levels up to a concentration of 7-KC of 20 μg/ml (Figure 5, A and C). Increasing doses of 7-KC also progressively impaired eNOS activity, and this effect was reversed by HDL (Figure 5D). These data demonstrate a strong correlation between decreased eNOS dimer levels and NOS activity in response to increasing doses of 7-KC and show that both effects are reversed by HDL. 7-KC did not affect inflammatory gene expression such as Il6 or Mcp1 (Supplemental Figure 2). Insig1 and LDL receptor mRNA levels, which are regulated by SREBP-2, were reduced by 7-KC (Supplemental Figure 2). The reduction of these mRNAs most likely reflected intracellular accumulation of 7-KC.
Effects of HDL on eNOS dimer, eNOS, and phospho-eNOS levels, and NOS activity in HAECs. HAECs were incubated with 7-KC (5–40 μg/ml) in the presence or absence of HDL (100 μg/ml) for 16 h, and cell lysates were analyzed by western blotting. (A) Western blot for eNOS dimer and monomer, total eNOS and phospho-eNOS. (B) Quantification of eNOS dimer/monomer levels. (C) Quantification of eNOS (filled bars) and phospho-eNOS (open bars). (D) NOS activity. The results are represented as mean ± SEM of 3 individual experiments. *P < 0.05 versus control.
Cholesterol and 7-KC mass efflux. Next, we measured cholesterol and 7-KC mass efflux to different acceptors in HAECs. HAECs were loaded with cholesterol (5 μg/ml) and 7-KC (5 μg/ml) for 24 h. Before starting efflux, intracellular cholesterol and 7-KC contents were measured to determine the baseline contents for the control dishes. Intracellular 7-KC contents were around 10 μg/mg protein and corresponded to the content in the isolated ECs from aortas in WTD-fed Abcg1–/– mice (Figure 4E).
HDL2 (25–100 μg/ml) and HDL3 (25–100 μg/ml) stimulated both cholesterol and 7-KC mass efflux, whereas apoA-I did not (Figure 6A). We examined the effect of suppression of ABCG1, ABCA1, and SR-BI by siRNA transfection on HDL-mediated cholesterol and 7-KC mass efflux. Western blotting showed effective suppression of ABCG1 and SR-BI; however, ABCA1 was not readily detected in these non–liver X receptor agonist–treated cells (Figure 6B, inset). Suppression of ABCG1 significantly reduced both cholesterol and 7-KC mass efflux (Figure 6B). Neither suppression of ABCA1 nor SR-BI affected cholesterol or 7-KC mass efflux (Figure 6B). These data indicate that the ABCG1-mediated sterol efflux pathway is predominant in HAECs.
Effects of ABCG1 and HDL on sterol efflux in HAECs. (A) HAECs were loaded with cholesterol or 7-KC mixture (each 5 μg/ml) for 24 h. HAECs were washed with PBS and incubated with different acceptors for 16 h. (B) HAECs were transfected with scrambled, ABCG1, ABCA1, or SR-BI siRNA. Twenty-four hours after transfection, HAECs were incubated with cholesterol (5 μg/ml) or 7-KC (5 μg/ml) for 24 h. HAECs were then washed with PBS and incubated with or without HDL2 (50 μg/ml) for 16 h. Results are represented as mean ± SEM of 3 individual experiments. Inset: Western blot for ABCG1 and SR-BI.
Effects of ABCG1 and HDL on eNOS dimer levels. We examined the effects of different concentrations of HDL on eNOS dimer disruption by 7-KC. HDL treatment protected the disruption of eNOS dimer levels in a concentration-dependent manner with concentrations between 25 and 100 μg/ml (Figure 7, A and B). The reduction of eNOS by 7-KC required a relatively long incubation time (>4 h) (Figure 7, C and D). We have previously reported a specific role of ABCG1 in the efflux of 7-oxysterols (35). To further evaluate the role of ABCG1 on eNOS dimer levels, we tested the effects of different oxysterols and cholesterol (each 10 μg/ml) in similar experiments. 7β-Hydroxycholesterol as well as 7-KC significantly decreased eNOS dimer levels (Figure 7, E and F). HDL treatment prevented 7β-hydroxycholesterol–induced eNOS dimer disruption. Cholesterol, 7α-hydroxycholesterol, 25-hydroxycholesterol, or 27-hydroxycholesterol did not affect eNOS dimer levels (Figure 7, E and F). This pattern of predominant effects of 7-oxysterols on eNOS dimer levels parallels the specific role of ABCG1 in promoting efflux of these oxysterols compared with other sterols (35).
Effects of HDL concentrations, incubation time with 7-KC, different oxysterols, and ABCG1 expression on eNOS dimer levels. (A and B) Effects of HDL concentrations on eNOS dimer disruption by 7-KC. HAECs were incubated with 7-KC (10 μg/ml) and HDL (25–200 μg/ml) for 16 h. (A) Western blot for eNOS dimer and monomer. (B) Quantification of the eNOS dimer/monomer. (C and D) Effects of incubation time with 7-KC on eNOS dimer disruption. (C) Western blot for eNOS dimer and monomer. (D) Quantification of the eNOS dimer/monomer. *P < 0.05 compared with no 7-KC at same time point. (E and F) Effects of different oxysterols on eNOS dimer disruption. HAECs were incubated with 10 μg/ml cholesterol or oxysterols in the presence or absence of HDL (100 μg/ml) for 16 h. 7αOH, 7α-hydroxycholesterol; 7βOH, 7β-hydroxycholesterol; 25OH, 25-hydroxycholesterol; 27OH, 27-hydroxycholesterol. (E) Western blot for eNOS dimer and monomer. (F) Quantification of the eNOS dimer/monomer. (G and H) HAECs were transfected with scrambled, ABCG1, ABCA1, or SR-BI siRNA. Forty-eight hours after transfection, HAECs were incubated with 7-KC (10 μg/ml) in the presence or absence of HDL (100 μg/ml) for 16 h. (G) Western blot for eNOS dimer and monomer. (H) Quantification of the eNOS dimer/monomer. The results are represented as mean ± SEM of 3 individual experiments. *P < 0.05 versus control.
To assess the specific role of ABCG1 in 7-KC–induced disruption of eNOS dimer levels, we knocked down expression of ABCG1 by siRNA. In ABCG1 siRNA-transfected HAECs, the protective effect of HDL was abolished (Figure 7, G and H). In contrast, suppression of neither ABCA1 nor SR-BI affected the ability of HDL to protect against disruption of eNOS dimer levels by 7-KC (Figure 7, G and H). These experiments show a specific requirement for ABCG1 in the ability of HDL to promote 7-KC efflux and to protect ECs from eNOS dimer disruption induced by 7-KC.
Effects of ABCG1 and HDL on ROS production. Previous studies have shown that disruption of eNOS dimer levels can be mediated by peroxynitrite (ONOO–), which is generated from superoxide (O2–) and NO (38). To investigate the hypothesis that HDL and ABCG1 reverse the effects of 7-KC on ROS production, we used the cell-permeable reagent 6-carboxy-2,7-dichlorodihydrofluorescein diacetate, diacetoxymethyl-ester (CM-H2DCFDA) (39). In HAECs, 7-KC (5–40 μg/ml) induced ROS formation in a dose-dependent manner (Figure 8, A and B). The ROS production by 7-KC required more than 4 h incubation (Figure 8C). The concentration dependence and the time-course response paralleled those of eNOS dimer disruption induced by 7-KC (Figure 7, A–D). 7β-Hydroxycholesterol (10 μg/ml) also significantly increased ROS (P < 0.01), whereas cholesterol, 7α-hydroxycholesterol, 25-hydroxycholesterol, or 27-hydroxycholesterol did not (data not shown). Incubation with HDL (100 μg/ml) significantly reduced ROS production by 7-KC (10 μg/ml) (Figure 8, D and E). This HDL protection was virtually abolished by ABCG1 knockdown (Figure 8, D and E). Similar protective effects of HDL and ABCG1 on ROS production by 7-KC were observed in primary mouse aortic ECs isolated in a manner similar to the studies described in Figure 4, D–F (see also Supplemental Figure 3). We also measured NOS activity in the similar experiments. HDL preserved the reduction of NOS activity by 7-KC, whereas the HDL protection was abolished by ABCG1 siRNA transfection (Figure 8F). These experiments suggest HDL preserves eNOS dimer levels and activity by promoting efflux of 7-KC via ABCG1 and thus reducing ROS formation.
Effects of ABCG1 and HDL in ROS production by 7-KC. (A and B) HAECs were incubated with 7-KC (1–40 μg/ml) for 16 h. Intracellular ROS was determined after 30 min of pulse, using CM-H2DCFDA. (A) Fluorescence of CM-H2DCFDA in HAECs. (B) Quantification of CM-H2DCFDA fluorescence. (C) Fold increase in fluorescence over time with 7-KC treatment. *P < 0.05 compared with no 7-KC at same time point. (D–F) HAECs were transfected with scrambled or ABCG1 siRNA. Forty-eight hours after transfection, HAECs were treated with 7-KC (10 μg/ml) in the presence or absence of HDL (100 μg/ml) for 16 h. (D) Fluorescence of CM-H2DCFDA. (E) Quantification of CM-H2DCFDA fluorescence. Inset: Western blot for ABCG1. (F) NOS activity. The results are represented as mean ± SEM of 3 individual experiments. Original magnification, ×200. *P < 0.05 versus control.
Effects of antioxidants and NG-nitro-l-arginine methyl ester on eNOS dimer levels. To confirm that 7-KC–induced eNOS dimer disruption is mediated by ROS, we also evaluated the effects of 2 potent antioxidants, glutathione (GSH) and N-acetylcysteine (NAC). Both GSH and NAC showed protective effects on eNOS dimer disruption (Figure 9, A and B) and ROS production (Figure 9, C and D). To determine the site of 7-KC–induced intracellular ROS production, HAECs were also incubated with carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation that abolishes the mitochondrial membrane proton gradient. CCCP did block the ability of 7-KC to induce ROS, suggesting that mitochondria were the source of ROS production by 7-KC (Supplemental Figure 4). By contrast, knockdown of NADPH oxidases Nox1, Nox2, and Nox4, did not affect 7-KC–induced ROS production (data not shown).
Effects of antioxidants and NOS inhibitor on eNOS dimer disruption by 7-KC. (A–D) HAECs were incubated with 7-KC (10 μg/ml) in the presence of GSH (10 mM), NAC (10 mM), or HDL (100 μg/ml) for 16 h. (A) Western blot for eNOS dimer and monomer. (B) Quantification of the eNOS dimer/monomer ratio. (C) Fluorescence of CM-H2DCFDA. Original magnification, ×200. (D) Quantification of CM-H2DCFDA fluorescence. (E and F) HAECs were incubated with 7-KC in the presence or absence of l-NAME for 16 h. (E) Western blot for eNOS dimer and monomer. (F) Quantification of the eNOS dimer/monomer ratio. (G) HAECs were incubated with 7-KC (5–20 μg/ml) in the presence of l-NAME or HDL (100 μg/ml) for 16 h. Western blot for nitrotyrosine. All lanes were run on the same gel but were noncontiguous between 7-KC and 7-KC + l-NAME. The results are represented as mean ± SEM of 3 individual experiments. *P < 0.05 versus vehicle control; #P < 0.05 versus 7-KC alone. Veh, vehicle.
To further analyze the mechanism of eNOS dimer disruption by 7-KC, we investigated the effect of NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME). l-NAME treatment prevented eNOS dimer disruption by 7-KC in a dose-dependent manner (Figure 9, E and F). We also investigated the effect of 7-KC on protein tyrosine nitrosylation, since nitrotyrosine formation is considered an indicator for ONOO– production. Treatment with 7-KC significantly increased the detection of nitrotyrosine-positive protein (Figure 9G). In addition, either the presence of l-NAME or HDL significantly reduced the level of nitrotyrosine formation (Figure 9G). These data strongly suggest that 7-KC induces formation of O2–, which reacts with eNOS-generated NO to form ONOO–, which in turn leads to eNOS oxidation.
Effect of apoA-I transgene expression in endothelial function. To further evaluate the role of HDL in endothelial function, we also investigated the effect of apoA-I transgene expression on endothelium-dependent vasorelaxation in HCD-fed Ldlr+/– mice. ApoA-I transgene expression significantly improved endothelium-dependent vasorelaxation (EC50: Ldlr+/–, 84.4 ± 11.6 vs. Ldlr+/–apoA-I Tg, 23.4 ± 6.5 nM; P < 0.05) (Figure 10A). There was no difference between the groups in the response to SNP (Figure 10B). ApoA-I transgene expression also significantly increased eNOS dimer levels (Figure 10, C and D) and NOS activity (Figure 10F). There was no difference between the groups in eNOS and phospho-eNOS levels (Figure 10, C and E). We also measured cholesterol and 7-KC contents in the aortas. In Ldlr+/–apoA-I Tg mice, both cholesterol (Figure 10G) and 7-KC (Figure 10H) contents were significantly decreased, but the magnitude of 7-KC reduction was more pronounced. These data suggest that increased HDL levels resulting from apoA-I transgene expression promote efflux of 7-KC from the aorta, contributing to preservation of eNOS dimer levels and activity.
Effect of apoA-I transgene expression in endothelial function in HCD-fed Ldlr+/– mice. Ldlr+/– and Ldlr+/–apoA-I Tg mice (n = 6 per group) were put on a HCD for 6 weeks. (A) ACh-induced vasorelaxation. (B) SNP-induced vasorelaxation. (C) Western blot for eNOS dimer and monomer, eNOS, and phospho-eNOS in aorta. (D) Quantification of the eNOS dimer/monomer ratio. (E) Quantification of eNOS and phospho-eNOS. (F) Aortic NOS activity. (G) Cholesterol and (H) 7-KC contents in aorta. The results are represented as mean ± SEM. *P < 0.05 versus control.
One of the most important athero-protective functions of HDL is thought to be the stimulation of macrophage cholesterol efflux, and recent studies have highlighted the key roles of ABCA1 and ABCG1 in reversing macrophage foam cell formation (40) and atherosclerosis (41, 42). HDL has also been shown to exert a variety of beneficial actions that are independent of macrophage cholesterol efflux. For example, HDL inhibits LDL oxidation, smooth muscle cell migration, and platelet aggregation and reverses endothelial dysfunction (20–22). Our studies revealed a non-redundant role of ABCG1 and a lesser role of ABCA1 in preserving endothelial eNOS activity in mice fed HCDs, and our results suggest that this may be a major mechanism underlying the ability of HDL to defend endothelial NO activity in response to such diets. The ability of ABCG1 to preserve endothelial function appears to be at least partly related to its role in promoting efflux of 7-oxysterols such as 7-KC to HDL.
HDL has consistently been shown to increase eNOS-dependent NO activity in cultured ECs (24, 25) and aortic rings (43) and in human forearm blood flow studies (44, 45). In humans, HDL levels are correlated with flow-mediated vasodilation responses of the brachial artery (18, 19) and with decreased coronary vasoconstrictor responses (44). Importantly, infusion of recombinant phospholipid/apoA-I particles into Tangier disease heterozygotes with isolated low HDL levels reversed defective forearm blood flow measurements (46). Deckert et al. (7, 8) showed that 7-oxysterols can produce decreased eNOS activity in rabbits and HUVECs. In apoE–/– mice fed a chow diet, arterial eNOS activity was preserved but became impaired when mice were challenged with a HCD (4). Importantly, apoA-I transgene expression reversed the decrease in eNOS activity induced by the HCD (4). The current study in HCD-fed Ldlr+/– mice reproduced preservation of endothelium-dependent aortic relaxation by apoA-I transgene overexpression (Figure 10), and we show that this effect is associated with preservation of eNOS dimer levels and eNOS activity and reduced aortic 7-KC levels.
Our studies extend these important earlier observations (4, 7, 8) and suggest that the underlying mechanism by which increased or basal HDL levels protect the endothelium involves efflux of dietary sterols, especially 7-oxysterols from ECs to HDL, mediated principally by ABCG1 (Figure 11). It is most likely that dietary oxysterols are normally incorporated into chylomicrons, cleared by the liver, converted into bile acids, and excreted (13). However, when there is delayed clearance of chylomicron remnants, as occurs in apoE–/– mice or in humans with increased coronary heart disease risk (47), the vascular endothelium has increased exposure to dietary oxysterols, and ABCG1 and HDL likely have a key role in excluding or promoting efflux of 7-oxysterols from ECs. Indeed, we found that ABCG1 was expressed specifically in endothelium in non-atherosclerotic mouse aorta (Figure 4A) and 7-KC also accumulated in ECs isolated from the aorta in Abcg1–/– mice (Figure 4E). Even though HDL may have a variety of different antioxidant properties in different settings (48, 49), the ability of HDL to promote efflux of 7-KC, reduce ROS production, and preserve eNOS dimer levels and activity were all dependent on ABCG1 expression, indicating that the underlying mechanism involves ABCG1-mediated oxysterol efflux. Notably, while 7-KC was readily detected in non-lesioned arteries from mice fed HCDs, it was not measurable in arteries from mice fed chow diets (Figure 2), making it unlikely that 7-KC was artifactually formed during sample processing. Moreover, 7-KC was specifically increased as a result of ABCG1 deficiency (Figure 2), consistent with the role of ABCG1 and not ABCA1 in promoting efflux of this oxysterol to HDL (35).
Diagram illustrating the sequence of events triggered by 7-KC and involved in eNOS dimer disruption and the inhibitory effect of HDL and ABCG1. CM, chylomicron; oxLDL, oxidized LDL.
In the present study in HAECs, disruption of eNOS dimer levels was induced by a 7-KC concentration of 5 μg/ml, which might be equivalent to levels found in human plasma after a fat-rich meal (50). Intracellular 7-KC content in HAECs treated with the relevant concentrations of 7-KC (5–10 μg/ml) were around 10 μg/mg protein, approximating the concentration found in isolated ECs from aortas in WTD-fed Abcg1–/– mice (Figure 4), in which eNOS dimer levels were reduced (Figure 3). Thus, these 7-KC concentrations are likely sufficient to induce endothelial dysfunction. The current findings agree with the notion that endothelial dysfunction is a key feature of early atherosclerosis (1) and also occurs transiently in the postprandial state (51).
Our parallel studies in mice and in HAECs suggest that ABCG1 mediates the efflux of 7-oxysterols from ECs to HDL, resulting in decreased ROS formation and preservation of the active dimeric form of eNOS (Figure 9). O2– is known to inactivate NO and generate ONOO– (37, 38). ONOO– can disrupt eNOS dimers through oxidation and displacement of the zinc metal ion (37, 52). Our studies also demonstrate that both l-NAME and antioxidants reversed the disruption of eNOS dimer levels by 7-KC (Figure 9). These data strongly suggest that 7-KC induced O2– and ONOO– production through interaction with NO, resulting in eNOS oxidation (Figure 11). There is considerable evidence that increased ROS can inhibit eNOS dimer formation and produce endothelial dysfunction in vivo, for example in diet-induced diabetic mice (53, 54) or in apoptosis signal–regulating kinase-1–deficient mice (55).
A number of different mechanisms have been proposed to account for the ability of HDL to preserve or increase arterial eNOS activity. HDL appears to be moderately effective in inducing eNOS-dependent vascular relaxation when directly added to aortic rings isolated from rats or mice (43, 56). However, the effect is very rapid (within a few minutes) and is saturated at very low concentrations of HDL (10 μg/ml), far below that normally bathing the endothelium (43). The response to added HDL is defective in vascular rings isolated from chow diet–fed SR-BI–/– mice (43), and from mice lacking the lysophospholipid S1P3 receptor (56). The direct effect of HDL on induction of eNOS activity has also been attributed to minor components such as lysophospholipids (56) or estrogen (57), but the concentrations of these components may not be sufficiently high to be physiologically relevant (25). While SR-BI may not have a major role in mediating net cellular cholesterol efflux to HDL in vivo, it is likely that ABCA1 and ABCG1 do mediate net efflux (27, 42, 58). Finally, our study has not assessed the role of ABC transporters in efflux to HDL of oxidized phospholipids, which are also likely to be important in endothelial dysfunction (59–61). Further studies are required to assess the relative roles of these different potential mechanisms in HDL-induced eNOS activity in vivo. Our study conclusively demonstrates the essential role of the ABC transporters, and especially ABCG1, in this process and delineates one mechanism involving efflux of 7-oxysterols and preservation of eNOS dimer levels.
Therapies that increase HDL levels, such as niacin and cholesteryl ester transfer protein inhibitors, probably activate the ABCG1-cholesterol/oxysterol efflux pathway not only in macrophages (26–28, 35) but also in ECs, likely with beneficial effects on endothelial function. Importantly, niacin therapy has been shown to improve NO-mediated vascular relaxation in humans (62). Our studies suggest that the underlying mechanism may involve increased efflux of cholesterol and 7-oxysterols via the ABCA1 and ABCG1 pathway.
Materials. The ROS-sensitive fluorescent probe CM-H2DCFDA and nuclear fast red were from Invitrogen. Anti-eNOS and anti–phospho-eNOS (S1177) antibodies were obtained from BD Transduction Laboratories. Anti-ABCG1, anti-PECAM, and anti-nitrotyrosine antibodies were purchased from Abcam. SR-BI antibody was from Santa Cruz Biotechnology Inc. Anti–β-actin antibody, X-gal (5-bromo-4-chloro-3-indolyl β-d-galactopyranoside), lipoprotein-deficient serum, NAC, GSH, phenylephrine, ACh, SNP, cholesterol, 7-KC, 7β-hydroxycholesterol, and 25-hydroxycholesterol were purchased from Sigma-Aldrich. 27-Hydroxycholesterol was obtained from Steraloids. l-NAME was purchased from Cayman Chemical. Human apoA-I was obtained from Biodesign International. HDL (density 1.063–1.210 g/ml), HDL2 (density 1.063–1.125 g/ml), and HDL3 (density 1.125–1.210 g/ml) were isolated by preparative ultracentrifugation from normolipidemic human plasma and stored in PBS.
Mouse studies. Abcg1–/–, Abca1–/–, and Abca1–/–Abcg1–/– mice have been previously described (41). We performed studies with a chow diet (0.025% cholesterol), a HCD (1.25% cholesterol, 7.5% cocoa butter, and 0.5% sodium cholate; catalog no. TD88051; Harlan Teklad) and a WTD (21% milk fat, 0.2% cholesterol; catalog no. TD88137; Harlan Teklad).
C57BL/6 Ldlr–/– mice and C57BL/6 apoA-I Tg mice (63) were obtained from the Jackson Laboratory and crossed to generate Ldlr+/–apoA-I Tg mice. Next, these animals were crossed with DBA/1LacJ mice (The Jackson Laboratory) to obtain the genetically uniform F1 generation. F1 hybrid C57BL/6 × DBA Ldlr+/–apoA-I Tg mice were put on the HCD.
Animals had ad libitum access to both food and water. Animal protocols were approved by the Institutional Animal Care and Use Committee of Columbia University.
Tissue collection. Mice were anesthetized with an intraperitoneal injection of ketamine. The chest and peritoneal cavity were opened and the circulatory system was perfused via the left ventricle with PBS. Aortas were removed and processed for all assays. For vascular studies, the left superficial femoral artery was removed and immediately placed in ice-cold physiologic salt solution.
Vascular function studies. Femoral arteries with intact endothelium and similar dimensions were mounted on a small vessel wire myograph (Danish MyoTechnology) as described previously (53). Vessels were bathed in physiologic salt solution at 37°C and aerated continuously with 5% CO2/95% O2 to achieve pH 7.4. The startup protocol and evaluation of vessel viability was conducted as described previously (53). Concentration response curves were performed for ACh (endothelium dependent) and SNP (endothelium-independent NO-releasing agent). Wall tension was expressed as mN/mm of artery length. Sensitivity to the agonist was expressed as the negative log of EC50 (–log EC50). Sensitivity was calculated from each concentration response curve by fitting the Hill equation using Prism (GraphPad Software).
Isolation of ECs from aorta. Mice aortas were perfused with PBS and digested in RPMI 1640 medium containing collagenase D (2 mg/ml; Roche Applied Science) at 37°C for 45 min. The digest was sequentially filtered through 100-μm, 70-μm, and 40-μm cell strainers and was washed with PBS. The cells were incubated with anti-PECAM biotin-conjugated antibody (Millipore) at 4°C for 15 min and were washed with PBS. Next, the cells were labeled with streptavidin microbeads (Miltenyi Biotec) and aortic ECs were separated by MACS column (Miltenyi Biotec) according to the manufacturer's instructions. Isolated aortic ECs were used for sterol mass measurement.
LacZ expression and PECAM immunostaining. The tissues were snap-frozen in OCT and stored at –80°C. Frozen sections 10 μm long were prepared. To determine β-galactosidase activity, the glass slides were incubated for 16 h in the presence of X-gal. The slides were counterstained with nuclear fast red. PECAM immunostaining was carried out as previously described (64).
Cell culture. HAECs and the culture medium EMG-2 were purchased from Lonza. The cells were grown in EMG-2 at 37°C in humidified 5% CO2 and used for experiments between passages 3 and 5. All siRNAs were purchased from Invitrogen or Santa Cruz Biotechnology Inc. HAECs were transfected with siRNA using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer's protocol. Forty-eight hours after transfection, HAECs were treated with 7-KC in the presence or absence of HDL.
Sterol mass analysis. The lipid fractions of abdominal aortas, isolated ECs or non-ECs from aorta, and HAECs were extracted using hexane/isopropanol (3:2 vol/vol) in presence of stigmasterol added as the internal standard. Total cholesterol and 7-KC were determined after saponification by gas-liquid chromatography (26, 35).
Sterol mass efflux assay. HAECs were incubated in EGM-2 plus 5% lipoprotein-deficient serum with cholesterol (5 μg/ml) and 7-KC (5 μg/ml) for 24 h. The next day, cells were washed with PBS and then incubated in EGM-2 plus 5% lipoprotein-deficient serum alone or supplemented with human apoA-I or HDL for 16 h. After the efflux period, media and cells were collected separately and lipids were extracted with hexane/isopropanol (3:2 vol/vol) with stigmastanol as the internal standard. Sterol mass of media and cells was determined using gas chromatography. Percentages of sterol mass efflux were calculated by the ratio of sterol mass in the medium to total (medium plus cellular) sterol mass.
NOS activity assay. The NO synthesizing activity was determined by quantifying the rate of the conversion of [3H]l-arginine to [3H]l-citrulline with kits obtained from Calbiochem-Novabiochem according to the manufacturer's instructions (52).
Western blotting. Protein was resolved on 4%–20% SDS-PAGE reducing gels (Bio-Rad). Protein was transferred to PVDF membranes and probed with primary antibodies overnight. For detection of eNOS dimer levels, we performed low-temperature SDS-PAGE (4°C) (52, 53). Mice aorta lysates and HAEC lysates were heated to 55°C and room temperature, respectively, for 30 min in the presence of SDS and 2.5% β-mercaptoethanol.
Quantification of intracellular ROS. The generation of intracellular ROS was estimated by incubating CM-H2DCFDA (1 μM) with cells 30 min before determination, as described previously (39).
Statistics. Statistical analysis was performed using the Student's t test. Bonferroni post-hoc tests were utilized. Results are represented as means ± SEM.
This work was supported by grants from the NIH (HL 54591).
Nonstandard abbreviations used: CM-H2DCFDA, 6-carboxy-2,7-dichlorodihydrofluorescein diacetate, diacetoxymethyl-ester; GSH, glutathione; HAEC, human aortic EC; HCD, high-cholesterol diet; 7-KC, 7-ketocholesterol; l-NAME, NG-nitro-l-arginine methyl ester; NAC, N-acetylcysteine; SNP, sodium nitroprusside; SR-BI, scavenger receptor B-I; WTD, Western diet.
Conflict of interest: A.R. Tall has received consulting fees from Pfizer, Merck, AstraZeneca, and Roche; lecture fees from Merck; and grant support from Merck and Pfizer.
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Shimokawa, H., Vanhoutte, P.M. 1989. Impaired endothelium-dependent relaxation to aggregating platelets and related vasoactive substances in porcine coronary arteries in hypercholesterolemia and atherosclerosis. Circ. Res. 64:900-914.
Deckert, V., et al. 1997. Inhibitors of arterial relaxation among components of human oxidized low-density lipoproteins. Cholesterol derivatives oxidized in position 7 are potent inhibitors of endothelium-dependent relaxation. Circulation. 95:723-731.
Deckert, V., Katan, M. 1998. Inhibition by cholesterol oxides of NO release from human vascular endothelial cells. Arterioscler. Thromb. Vasc. Biol. 18:1054-1060.
Brown, A.J., Jessup, W. 1999. Oxysterols and atherosclerosis. Atherosclerosis. 142:1-28.
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Zhou, Q., Wasowicz, E., Handler, B., Fleischer, L., Kummerow, F.A. 2000. An excess concentration of oxysterols in the plasma is cytotoxic to cultured endothelialcells. Atherosclerosis. 149:191-197.
Jacobson, M.S. 1987. Cholesterol oxides in Indian ghee: possible cause of unexplained high risk of atherosclerosis in Indian immigrant populations. Lancet. 2:656-658.
Vine, D.F., Mamo, J.C.L., Beilin, L.J., More, T.A., Croft, K.D. 1998. Dietary oxysterols are incorporated in plasma triglyceride-rich lipoproteins, increase their susceptibility to oxidation and increase aortic cholesterol concentration of rabbits. J. Lipid Res. 39:1995-2004.
Sander, B.D., Smith, D.E., Addis, P.B., Park, S.W. 1989. Effects of prolonged and adverse storage conditions on levels of cholesterol oxidation products in dairy products. J. Food Sci. 54:874-879.
van de Bovenkamp, P., Kosmeijer-Schuil, T.G., Katan, M.B. 1988. Quantification of oxysterols in Dutch foods: egg products and mixed diets. Lipids. 23:1079-1085.
Gordon, D.J., Rifkind, B.M. 1989. High-density lipoprotein-the clinical implications of recent studies. N. Engl. J. Med. 321:1311-1316.
Assmann, G., Gotto (Jr.), A.M. 2004. HDL cholesterol and protective factors in atherosclerosis. Circulation. 109(Suppl. 1):III-8-III-14.
Li, X.P., et al. 2000. Protective effect of high density lipoprotein on endothelium-dependent vasodilatation. Int. J. Cardiol. 73:231-236.
Kuvin, J.T., et al. 2003. Relation between high-density lipoprotein cholesterol and peripheral vasomotor function. Am. J. Cardiol. 92:275-279.
O'Connell, B.J., Genest (Jr.), J. 2001. High-density lipoproteins and endothelial function. Circulation. 104:1978-1983.
Rohrer, L., Hersberger, M., von Eckardstein, A. 2004. High density lipoproteins in the intersection of diabetes mellitus, inflammation and cardiovascular disease. Curr. Opin. Lipidol. 15:269-278.
Mineo, C., Deguchi, H., Griffin, J.H., Shaul, P.W. 2006. Endothelial and antithrombotic actions of HDL. Circ. Res. 98:1352-1364.
Collins, T., Cybulsky, M.I. 2001. NF-kappaB: pivotal mediator or innocent bystander in atherogenesis. ? J. Clin. Invest. 107:255-264.
Uittenbogaard, A., Shaul, P.W., Yuhanna, I.S., Blair, A., Smart, E.J. 2000. High density lipoprotein prevents oxidized low density lipoprotein-induced inhibition of endothelial nitric-oxide synthase localization and activation in caveolae. J. Biol. Chem. 275:11278-11283.
Matsuura, F., Wang, N., Chen, W., Jiang, X.C., Tall, A.R. 2006. HDL from CETP-deficient subjects shows enhanced ability to promote cholesterol efflux from macrophages in an apoE- and ABCG1-dependent pathway. J. Clin. Invest. 116:1435-1442.
Yvan-Charvet, L., et al. 2007. Inhibition of cholesteryl ester transfer protein by torcetrapib modestly increases macrophage cholesterol efflux to HDL. Arterioscler. Thromb. Vasc. Biol. 27:1132-1138.
Tall, A.R., Yvan-Charvet, L., Terasaka, N., Pagler, T., Wang, N. 2008. HDL, ABC transporters, and cholesterol efflux: implications for the treatment of atherosclerosis. Cell Metab. 7:365-375.
O'Connell, B.J., Denis, M., Genest, J. 2004. Cellular physiology of cholesterol efflux in vascular endothelial cells. Circulation. 110:2881-2888.
Wang, N., Silver, D.L., Costet, P., Tall, A.R. 2000. Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1. J. Biol. Chem. 275:33053-33058.
Oram, J.F., Lawn, R.M., Garvin, M.R., Wade, D.P. 2000. ABCA1 is the cAMP-inducible apolipoprotein receptor that mediates cholesterol secretion from macrophages. J. Biol. Chem. 275:34508-34511.
Wang, N., Lan, D., Chen, W., Matsuura, F., Tall, A.R. 2004. ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins. Proc. Natl. Acad. Sci. U. S. A. 101:9774-9779.
Kennedy, M.A., et al. 2005. ABCG1 has a critical role in mediating cholesterol efflux to HDL and preventing cellular lipid accumulation. Cell Metab. 1:121-131.
Vaughan, A.M., Oram, J.F. 2005. ABCG1 redistributes cell cholesterol to domains removable by high density lipoprotein but not by lipid-depleted apolipoproteins. J. Biol. Chem. 275:34508-34511.
Terasaka, N., Wang, N., Yvan-Charvet, L., Tall, A.R. 2007. High-density lipoprotein protects macrophages from oxidized low-density lipoprotein-induced apoptosis by promoting efflux of 7-ketocholesterol via ABCG1. Proc. Natl. Acad. Sci. U. S. A. 104:15093-15098.
Rodriguez-Crespo, I., Moenne-Loccoz, P., Loehr, T.M., Ortiz de Montellano, P.R. 1997. Endothelial nitric oxide synthase: modulations of the distal heme site produced by progressive N-terminal deletions. Biochemistry. 36:8530-8538.
Forstermann, U., Munzel, T. 2006. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation. 113:1708-1714.
Xu, J., Xie, Z., Reece, R., Pimental, D., Zou, M.H. 2006. Uncoupling of endothelial nitric oxidase synthase by hypochlorous acid: role of NAD(P)H oxidase-derived superoxide and peroxynitrite. Arterioscler. Thromb. Vasc. Biol. 26:2688-2695.
Robbesyn, F., et al. 2003. HDL counterbalance the proinflammatory effect of oxidized LDL by inhibiting intracellular reactive oxygen species rise, proteasome activation, and subsequent NF-kappaB activation in smooth muscle cells. FASEB J. 17:743-745.
Wang, X., et al. 2007. Macrophage ABCA1 and ABCG1, but not SR-BI, promote macrophage reverse cholesterol transport in vivo. J. Clin. Invest. 117:2216-2224.
Yvan-Charvet, L., et al. 2007. Combined deficiency of ABCA1 and ABCG1 promotes foam cell accumulation and accelerates atherosclerosis in mice. J. Clin. Invest. 117:3900-3908.
Rader, D.J. 2006. Molecular regulation of HDL metabolism and function: implications for novel therapies. J. Clin. Invest. 116:3090-3100.
Yuhanna, I.S., et al. 2001. High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase. Nat. Med. 7:853-857.
Zeiher, A.M., Schachlinger, V., Hohnloser, S.H., Saurbier, B., Just, H. 1994. Coronary atherosclerotic wall thickening and vascular reactivity in humans. Elevated high-density lipoprotein levels ameliorate abnormal vasoconstriction in early atherosclerosis. Circulation. 89:2525-2532.
Bisoendial, R.J., et al. 2003. Restoration of endothelial function by increasing high-density lipoprotein in subjects with isolated low high-density lipoprotein. Circulation. 107:2944-2948.
Bansal, S., et al. 2007. Fasting compared with nonfasting triglycerides and risk of cardiovascular events in women. JAMA. 298:309-316.
Nicholls, S.J., et al. 2005. Reconstituted high-density lipoproteins inhibit the acute pro-oxidant and proinflammatory vascular changes induced by a periarterial collar in normocholesterolemic rabbits. Circulation. 111:1543-1550.
Tall, A.R. 2008. Cholesterol efflux pathways and other potential mechanisms involved in the athero-protective effect of high density lipoproteins. J. Intern. Med. 263:256-273.
Emanuel, H.A., Hassel, C.A., Addis, P.B., Bergman, S.D., Zavoral, J.H. 1991. Plasma cholesterol oxidation products (oxysterols) in human subjects fed a meal rich in oxysterols. J. Food Sci. 56:843-847.
Vogel, R.A., Corretti, M.C., Plotnick, G.D. 1997. Effect of a single high-fat meal on endothelial function in healthy subjects. Am. J. Cardiol. 79:350-354.
Zou, M.H., Shi, C., Cohen, R.A. 2002. Oxidation of the zinc-thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite. J. Clin. Invest. 109:817-826.
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Hink, U., et al. 2001. Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ. Res. 88:E14-E22.
Yamashita, T., et al. 2007. Apoptosis signal-regulating kinase-1 is involved in vascular endothelial and cardiac remodeling caused by nitric oxide deficiency. Hypertension. 50:519-524.
Nofer, J.R., et al. 2004. HDL induces NO dependent vasorelaxation via the lysophospholipid receptor S1P3. J. Clin. Invest. 113:569-581.
Gong, M., et al. 2003. HDL-associated estradiol stimulates endothelial NO synthase and vasodilation in an SR-BI-dependent manner. J. Clin. Invest. 111:1579-1587.
Yvan-Charvet, L., et al. 2008. SR-BI inhibits ABCG1-stimulated net cholesterol efflux from cells to plasma HDL. J. Lipid Res. 49:107-114.
Watson, A.D., et al. 1997. Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induce monocyte/endothelial interactions and evidence for their presence in vivo. J. Biol. Chem. 272:13597-13607.
Rikitake, Y., et al. 2000. Inhibition of endothelium-dependent arterial relaxation by oxidized phosphatidylcholine. Atherosclerosis. 152:79-87.
Navab, M., et al. 2004. The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL. J. Lipid Res. 45:993-1007.
Kuvin, J.T., et al. 2002. A novel mechanism for the beneficial vascular effects of highdensity lipoprotein cholesterol: enhanced vasorelaxation and increased endothelial nitric oxide synthase expression. Am. Heart J. 144:165-172.
Rubin, E.M., Ishida, B.Y., Clift, S.M., Krauss, R.M. 1991. Expression of human apolipoprotein A-I in transgenic mice results in reduced plasma levels of murine apolipoprotein A-I and the appearance of two new high density lipoprotein size subclasses. Proc. Natl. Acad. Sci. U. S. A. 88:434-438.
Welch, C.L., et al. 2007. Spontaneous atherothrombosis and medial degradation in Apoe–/–, Npc1–/– mice. Circulation. 116:2444-2452.
Version 1 (October 16, 2008): No description
Version 2 (November 3, 2008): No description

redpajama_set_name: RedPajamaCommonCrawl


text:
Madagascar In the News
Background: Formerly an independent kingdom, Madagascar became a French colony in 1896 but regained independence in 1960. During 1992-93, free presidential and National Assembly elections were held ending 17 years of single-party rule. In 1997, in the second presidential race, Didier RATSIRAKA, the leader during the 1970s and 1980s, was returned to the presidency. The 2001 presidential election was contested between the followers of Didier RATSIRAKA and Marc RAVALOMANANA, nearly causing secession of half of the country. In April 2002, the High Constitutional Court announced RAVALOMANANA the winner. RAVALOMANANA achieved a second term following a landslide victory in the generally free and fair presidential elections of 2006. In early 2009, protests over increasing restrictions on opposition press and activities resulted in RAVALOMANANA stepping down and the presidency was conferred to the mayor of Antananarivo, Andry RAJOELINA. Following negotiations in July and August of 2009, a power-sharing agreement with a 15-month transitional period was established, but has not yet been implemented.
Location: Southern Africa, island in the Indian Ocean, east of Mozambique
Country name conventional long form: Republic of Madagascar
Country name conventional short form: Madagascar
Country name former: Malagasy Republic
Age structure: 0-14 years: 43.1% (male 4,762,589/female 4,693,259); 15-64 years: 53.8% (male 5,864,520/female 5,938,029); 65 years and over: 3% (male 295,409/female 372,415) (2011 est.);
Sex ratio: at birth: 1.03 male(s)/female; under 15 years: 1.01 male(s)/female; 15-64 years: 0.99 male(s)/female; 65 years and over: 0.8 male(s)/female; total population: 0.99 male(s)/female (2011 est.);
Infant mortality rate: total: 51.45 deaths/1,000 live births; male: 56.23 deaths/1,000 live births; female: 46.52 deaths/1,000 live births (2011 est.);
HIV/AIDS - deaths: 1,700 (2009 est.);
Nationality: noun: Malagasy (singular and plural); adjective: Malagasy;
Ethnic groups: Malayo-Indonesian (Merina and related Betsileo), Cotiers (mixed African, Malayo-Indonesian, and Arab ancestry - Betsimisaraka, Tsimihety, Antaisaka, Sakalava), French, Indian, Creole, Comoran;
Religions: indigenous beliefs 52%, Christian 41%, Muslim 7%;
Languages: French (official), Malagasy (official), English;
Literacy: definition: age 15 and over can read and write; total population: 68.9%; male: 75.5%; female: 62.5% (2003 est.);
GDP (official exchange rate): $8.33 billion (2010 est.);
GDP - real growth rate: 1.5% (2010 est.); -1% (2009 est.); 7% (2008 est.);
GDP - composition by sector: agriculture: 26.5%; industry: 16.7%; services: 56.8% (2010 est.);
Population below poverty line: 50% (2004 est.);
Labor force: 9.504 million (2007);
Budget: revenues: $896.9 million; expenditures: $1.547 billion (2010 est.);
Industries: meat processing, seafood, soap, breweries, tanneries, sugar, textiles, glassware, cement, automobile assembly plant, paper, petroleum, tourism;
Electricity - consumption: 971.4 million kWh (2007 est.);
Mad for Marc
Following a hotly-disputed election, Madagascar has two governments. Sarah Coleman reviews the regional press.


text:
Kristina Andreotta
Health care worker Demetra Ransom comforts a patient in the COVID-19 ward at United Memorial Medical Center in Houston last month. The U.S. has since surpassed 20 million confirmed coronavirus cases. Mark Felix/AFP via Getty Images hide caption toggle caption Mark Felix/AFP via Getty Images Health care worker Demetra Ransom comforts a patient in the…
CLOSE President-elect Joe Biden called the lack of cooperation from the Trump administration irresponsible. USA TODAYUSA TODAY is keeping track of the news surrounding COVID-19 as a pair of vaccines join the U.S. fight against a virus that has killed more than 335,000 Americans since the first reported fatality in February. Keep refreshing this page for…
News Jess Szabo - January 15, 2021
U.S. Senator Charles Schumer announced today that after numerous discussions with President-elect Joe Biden and his team, his incoming administration's FEMA will deliver roughly $2 billion MORE to New York State for COVID-19 relief. Schumer,...
NEW YORK – New York Attorney General Letitia James released the following statement on reforms to the tax lien sale: "Over the last few months, I have worked hard with stakeholders and lawmakers to address...
Utica community chooses logo for America's Greatest Heart Run & Walk 2021 Winning...
UTICA, January 14, 2021 – America's Greatest Heart Run & Walk was one of the area's last in-person events of 2020. We've all pivoted, and the work of the community continues – even if...
Congress Overturns Trump Veto On Defense Bill After Political Detour
Senate Majority Leader Mitch McConnell, R-Ky., at the Capitol on Wednesday. The Senate voted Friday to override President Trump's veto on the annual defense bill. Susan Walsh/AP hide caption toggle caption Susan Walsh/AP Senate Majority Leader Mitch McConnell, R-Ky., at the Capitol on Wednesday. The Senate voted Friday to override President Trump's veto on the…
Louisiana Congressman-elect Luke Letlow dies of COVID-19
Greg Hilburn, Monroe News-Star Published 10:02 p.m. ET Dec. 29, 2020 | Updated 11:07 a.m. ET Dec. 30, 2020CLOSE Luke Letlow, who won the 5th Congressional District seat in Louisiana, died with COVID-19. USA TODAYLouisiana Congressman-elect Luke Letlow died of COVID-19 on Tuesday at Ochsner LSU Health in Shreveport.Letlow, 41, was transferred from St. Francis Medical Center…
Utica Phoenix Radio Owner Debuts WNPR Game Show
News charrislockwood - January 14, 2021
By Jess Szabo CHL Newest Panelist on NPR's Says You! "A public radio game show of bluff and bluster, words and whimsy" On January 2, 2021, National Public Radio's famous talk show, "Says You" offered a special...
Attorney General James Ready to Take Legal Action Against Insurrectionists or Domestic Terrorists Who...
NEW YORK – New York Attorney General Letitia James today released the following statement in response to reports that insurrectionists and domestic terrorists are considering riots at the New York state Capitol in Albany...
Governor Cuomo Updates State on Covid-19 Fight
Governor Andrew M. Cuomo yesterday updated New Yorkers on the state's progress during the ongoing COVID-19 pandemic. Three additional cases of the UK variant have been identified in Warren County, bringing the total number...


text:
package com.jeff.miniflix.models;

import com.google.common.collect.ImmutableList;
import com.google.gson.JsonObject;

import java.sql.*;
import java.util.List;
import java.util.Optional;

public class Category extends Model {

    private static final String ID_NAME = "Name";

    public static Optional<JsonObject> getByName(String name) {
        Optional<JsonObject> catOptional = Optional.empty();
        Connection connection = null;
        PreparedStatement statement = null;
        ResultSet r = null;
        try {
            connection = connect();
            statement = connection.prepareStatement("SELECT ID, Name FROM Categories WHERE Name = ?");
            statement.setString(1, name);
            r = statement.executeQuery();
            if (r.next()) {
                catOptional = Optional.of(from(r));
            }
        } catch (SQLException e) {
            e.printStackTrace();
        } finally {
            close(connection, statement, r);
        }
        return catOptional;
    }

    public static List<JsonObject> getAll() {
        ImmutableList.Builder<JsonObject> array = new ImmutableList.Builder<>();
        Connection connection = null;
        Statement statement = null;
        ResultSet r = null;
        try {
            connection = connect();
            statement = connection.createStatement();
            r = statement.executeQuery("SELECT ID, Name FROM Categories");
            while (r.next()) {
                array.add(from(r));
            }
        } catch (SQLException e) {
            e.printStackTrace();
        } finally {
            close(connection, statement, r);
        }
        return array.build();
    }

    private static JsonObject from(ResultSet r) throws SQLException {
        JsonObject object = new JsonObject();
       // object.addProperty(r.getString(ID_ID), r.getString(ID_NAME));
        object.addProperty(ID_ID, r.getInt(ID_ID));
        object.addProperty(ID_NAME, r.getString(ID_NAME));
        return object;
    }

    /*public final int id;
    public final String name;

    public Category(int id, String name) {
        this.id = id;
        this.name = name;
    }

    @Override
    public JsonObject toJson() {
        JsonObject ob = new JsonObject();
        ob.addProperty(ID_ID, id);
        ob.addProperty(ID_NAME, name);
        return ob;
    }*/
}

redpajama_set_name: RedPajamaGithub


Britain's Simon Yates (Mitchelton-Scott) has become the new leader of the UCI WorldTour following his overall victory in the Vuelta Ciclista a España on Sunday.
Yates, 26, has ousted longstanding leader and reigning UCI World Champion Peter Sagan (BORA-hansgrohe) from the top spot overall, and is the first rider to amass more than 3000 UCI WorldTour points this season.
A winner of a stage as well as the leader of the Vuelta a España for 10 days, Yates has now garnered a total of 3072 points. He also took points in the Giro d'Italia, where he won three stages and led for 12 days, as well as in the Tour de Pologne, Volta a Catalunya and Paris-Nice.
Sagan drops to second place behind Yates with 2992 points, whilst Alejandro Valverde (Movistar Team), fifth in the Vuelta a España and the winner of two stages, ascends eight places in the ranking to third overall, with 2539 points. Enric Mas (Quick-Step Floors), who finished second in the Vuelta a España, has gained 41 spots and is now 24th overall in the UCI WorldTour ranking, whilst Miguel Angel Lopez (Astana Pro Team), repeated his third place this spring in the Giro d'Italia with third overall in the Vuelta, and has moved up to 13th.
Yates took a definitive lead on the Vuelta a España on stage 14 at Los Praeres, winning on the tough summit finish in northern Spain, then made a strong defence of his lead in the third week's main time trial at Torrelavega and the last two mountain stages in the Pyrenées. He concluded the Vuelta a España with a 1.46 advantage over Mas, and 2.04 lead on third-placed Lopez.
In the UCI WorldTour teams ranking, Quick-Step Floors, who netted four stages and a second place overall in the Vuelta a España, continue to lead the classification, as they have done since March. Mitchelton-Scott, for whom the Vuelta is their first ever Grand Tour victory, move up to fourth.
Simon Yates' victory in the Vuelta a España means Great Britain has become the first nation ever to secure victories in all three Grand Tours in a single season with three different riders. Chris Froome (Team Sky) won the Giro d'Italia and finished third in the Tour de France, won by his team-mate Geraint Thomas, whilst Yates (Mitchelton-Scott) rounded off Britain's extraordinary run of success with victory in the Vuelta.
Inspired by compatriot Thor Hushovd as a young racer, Norway's Amund Grondahl Jansen, like the former UCI World Champion, is a Classics racer and sprinter. Before joining his first UCI WorldTour Team Lotto NL-Jumbo, in 2017, Jansen (24) secured a string of top results in the U23 category. In 2016 alone, he triumphed in his country's National U23 road race, took a stage of the Tour de l'Avenir, and also captured two top stage races, the ZLM Roompot Tour in the Netherlands and the Tour de Gironde in France. He rounded off his season with fifth in the U23 World Championships road race.
This year, Jansen has completed his first ever Grand Tour, the Tour de France, in 139th, as a support rider and made big progress in his second Paris-Roubaix - which he once targeted as his dream race - in 16th. That was Jansen first top 20 in a Monument and a big advance on his 94th place in Paris-Roubaix in 2017.


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Look out for the page titled Tracking Code. This will display your unique tracking code. It will look something like: UA-1234567-01.
Enter your Storbie shop's Manage area.
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text:
Scrutable
Open to critical enquiry
Board index Scrutable Weighty Matters
Edward Colston statue pulled down
Discussions about serious topics, for serious people
dyqik
Light of Blast
Location: Masshole
Contact dyqik
AOL Twitter
Re: Edward Colston statue pulled down
Post by dyqik » Fri Jan 07, 2022 2:55 pm
lpm wrote: ↑
Woodchopper wrote: ↑
I don't think that the verdict is perverse.
But the protestors clearly got off because the jury sympathized with their cause. I'm fine with that as one of the reasons for having trial by jury is that in exceptional circumstances people can be acquitted even though they have clearly committed a crime. Juries are supposed to be able to bring in what they consider to be natural justice rather than administer the law.
Er... that's the very definition of a perverse verdict.
It's when a jury reckons the defendant is guilty per the letter of the law, but considers the spirit of the law or natural justice to be more valuable than the letter of the law.
The letter of the law includes the exceptions to the law given by common law and the other considerations the Secret Barrister lists. No law stands alone, or can be viewed in isolation.
Dorkwood
Post by monkey » Fri Jan 07, 2022 3:17 pm
dyqik wrote: ↑
Which is why Woodchopper was wrong when he said "clearly committed a crime". As far as I understand it, a perverse verdict would be when the law doesn't allow the argued exception, or the jury believes the defendant didn't meet the criteria for them, but acquits anyway. That's not what happened here.
The defendants clearly chucked a statue in the river, but it was not clear that this was criminal.
Woodchopper
Post by Woodchopper » Fri Jan 07, 2022 3:26 pm
monkey wrote: ↑
Yes, I was thinking of 'perverse' in its general meaning (unreasonable or unacceptable) rather than the specific meaning that you and lpm refer to.
Stummy Beige
Post by lpm » Fri Jan 07, 2022 3:46 pm
I think we've very very very safe in assuming the jury didn't actually believe the defendants believed chucking a statue in the river would increase its value. And didn't actually believe the defendants believed the statue was an indecent display. And didn't actually believe the defendants believed the statue was about to commit a crime like a Hawk fighter about to murder children in East Timor. And didn't actually believe the defendants believed they owned the statue. And didn't actually believe the defendants believed the owners of the statue wanted it chucked in the harbour.
And I don't think any of you actually believe the jury actually believed the defendants believed any of these things either. Due to the fact that, you know, not all of you are complete morons.
Everyone in the game is just pretending to believe things they don't actually believe in order to get the outcome we want.
Awarded gold star 4 November 2021
@Woodchopper, It's called jury nullification in the US, which seems harder to less specific about.
Also, I agreed with your general point - Laws should have exceptions and thresholds, and juries should be able to acquit for whatever reason they see fit, as the alternative would be worse.
The wiki is quite interesting on the history. I'd forgotten the Clive Ponting case.
https://en.wikipedia.org/wiki/Jury_nullification
Bird on a Fire
Princess POW
Post by Bird on a Fire » Fri Jan 07, 2022 4:09 pm
Surely by the standards of today a statue of a racist human trafficker is indecent? While the word makes me think of pearl-clutching old folks tutting about miniskirts, it's clearly meant to refer to things that would cause shock/outrage/upset in a public place, which the Colston statue evidently had for decades. Not much of a stretch to understand why it could be considered abusive either.
I expect expert witness Professor David Olusoga was eloquently persuasive when he argued that point.
As for owner's consent - it's a public statue, owned by the public, and the public wanted it down. The council were dragging their heels, but that doesn't mean people can't help themselves. If I fix a park bench or fill in a pothole it wouldn't be criminal damage either.
You can shove your climate crisis up your arse!
Take thy beak from out my heart, and take thy form from off my door.
noggins
Fuzzable
Post by noggins » Fri Jan 07, 2022 4:16 pm
Funny thing is, tory ire at this sort of thing is going to egg juries on to be perverse.
Yes, and you only need 3 dissenters for a hung jury, so even a few perverse jurors can make a difference.
You seem to be positing that the jury are all morons, and couldn't understand the legal arguments put before them, or evaluate them objectively as they were sworn to do. That's a massive piece of prejudice on your part.
JQH
After Pie
Location: Sar Flandan
Post by JQH » Fri Jan 07, 2022 5:24 pm
Atilla the Stockbroker made the interesting point that the people whinging about the statue of Colston being pulled down were more than happy to see statues of Stalin pulled down in 1989 (and statues of Saddam Hussein in 2003).
And remember that if you botch the exit, the carnival of reaction may be coming to a town near you.
Fintan O'Toole
JQH wrote: ↑
They seem oddly quiet about the Nursultan Nazarbaye one yesterday though.
You muppet. I'm positing the opposite. That the jury understood the legal arguments, rejected them for being silly and delivered the correct verdict nonetheless.
You, on the other hand, seem to be prejudiced against these jurors and think they're morons who believed stuff that was barely delivered with a straight face.
Oh, ok. You're positing that the defense lawyers and judge don't take the right to protest, whether a prosecution is proportionate, or the other laws that the defense raised seriously.
Perhaps you're too devoted to science and the more orderly processes there?
The defense lawyers play with home advantage - and don't need to play fair in the same way that prosecutors do. As Disco said, why not throw stuff out there and see if the jury wants to pick it up. A jury trial is not about ordered legal rationality.
wilsontown
Stargoon
Joined: Tue Feb 23, 2021 11:51 am
Post by wilsontown » Fri Jan 07, 2022 6:38 pm
I mean, we literally do not know anything about why the jury came to the conclusion it did. So erecting narratives about whether or not they're a bunch of morons seems a pointless exercise.
"All models are wrong but some are useful" - George Box
We can apply the man on the Clapham omnibus test. Simply assume the jurors are ordinary and reasonable people, reasonably educated and intelligent but nondescript.
Which leads us to expect the jurors weren't morons believing silly stuff about indecency, but ordinary people who appreciate the value of justice.
Is it just me who thinks the defence made some good points then?
I'm sure we can all pretend to each other the defense made some good points.
The real test comes if we still think they're good points if applied by Tory scum to things we like.
But the specifics of the case are so unusual I'm really not convinced they could easily be applied elsewhere.
There are not many things similar to slavery. It's universally recognised as abhorrent and has been illegal for nearly 200 years. It also had millions of victims, many of whose descendents now live in the UK.
There are also not many things similar to racism.
I think you'll struggle to come up with an example of anything "we like" that has the relevant characteristics.
This case certainly doesn't set any kind of precedent for destroying statues/art in a general sense. It will be setting precedents for how the UK deals with the legacy of slavery.
El Pollo Diablo
Location: FBPE
Post by El Pollo Diablo » Fri Jan 07, 2022 8:32 pm
Vigilante manslaughter/murder of a paedophile, for instance, might do it.
Why you gotta act like you know when you don't know?
It's okay if you don't know everything
- Ben Folds
Which of the defence's arguments could be used for a murder or manslaughter charge?
I think the list of exceptions for killing a person is probably a bit different to the list of exceptions for committing criminal damage.
Fishnut
Post by Fishnut » Fri Jan 07, 2022 9:05 pm
The 'technically guilty but morally innocent' have been acquitted before. BoaF has already linked to these articles and I linked to this collection. To summarise for those who avoid links (in chronological order):
The Ploughshare Four - Angie Zelter, Joanna Wilson, Lotta Kronlid, and Andrea Needham - used hammers to cause £1.5m of damage to a recently built multi-purpose Hawk which has been destined for Indonesia where it was most likely to be used against civilians in East Timor. They claimed their actions were reasonable under the Genocide Act and because they used reasonable force to prevent a crime. The jury found them not guilty. Three years later the British government admitted that "British-made jets were being used by the Indonesians to intimidate the people of East Timor".
BoaF's article ends by saying that "The judgment.. will have repercussions in international jurisprudence and diplomacy". Yet this piece from 2017 notes that,
20 years on from the Ploughshares case, Britain is still very successful at selling arms abroad. In the latest figures published by the Department of Trade and Industry, the UK is said, to be the second largest global defence exporter on a rolling ten-year basis.
It seems those 'repercussions' were limited.
Rosie James and Rachel Wenham swam to a Trident submarine docked in Barrow-in-Furness, painted "illegal" and "Death Machine" on it, put a banner saying "Women Want Peace" over the conning tower and damaged equipment. They were accused of causing £25,000 of criminal damage. The first trial was halted because of screw-ups by the prosecution (my editorialising). The second trial found them not guilty of criminal damage but couldn't come to an agreement on the main charge of disarming radar-testing equipment.
The jury were only allowed to consider the very narrow grounds of defence of necessity and self defence allowed under English law, the judge having ruled out defences based on international law... [making] the extraordinary statement that "crimes against humanity are not crimes in English law
A third trial followed and the jury, again, were unable to reach a verdict.
(Scotland) - Angie Zelter, Ellen Moxley and Ulla Roder were charged with causing £80,000 of damage to a Trident-related acoustic barge. They argued that they had exercised their legal right to prevent a crime as Trident could not be used in a way that was lawful under international law, and that while their actions were "willful, they had not been malicious". The sheriff agreed and instructed the jury to acquit the women, which they did. Interestingly, the Lord Advocate said he intended to refer the case to the Court of Criminal Appeal due to the decision, in a move reminiscent of that by the Attorney General today. The clarification can be seen here (I haven't read it, it's really long!).
Sylvia Boyes and River entered a dockyard with the intention of disarming a Trident submarine with an axe. "They denied criminal damage, claiming their actions were justifed because nuclear weapons were immoral and illegal under international law." The judge told the jury that "ideals formed no defence against a criminal damage charge" yet they decided to acquit them anyway.
Lord Melchett (executive director of Greenpeace, not the one from Blackadder) and 27 other protestors admitted destroying GM crops "but said that acting to prevent pollen from the genetically-modified maize from polluting neighbouring organic crops and gardens". The jury acquitted them.
Toby Olditch and Philip Pritchard broke into an RAF site in 2003 - the day before bombing in Iraq began - with the intention of clogging the engines of US B52 bombers. They pleaded "not guilty... to conspiring to cause criminal damage, claiming the B52s would have been used to commit war crimes in Iraq." They had been tried the year before but the jury failed to reach a verdict. The second time round the jury acquitted them of all charges.
Ben Stewart, Will Rose, Kevin Drake, Tim Hewke, Huw Williams and Emily Hall were accused of causing £30,0000 of criminal damage at a coal-fired power station.
The activists admitted trying to shut down the station by occupying the smokestack and painting the word "Gordon" down the chimney, but argued that they were legally justified because they were trying to prevent climate change causing greater damage to property around the world. It was the first case in which preventing property damage caused by climate change had been used as part of a "lawful excuse" defence in court.
The jury acquitted them by a majority verdict.
Rev Daniel Woodhouse and Samuel Walton broke into site owned by BAE systems with the intention of disarming Tornado jets being used by Saudi Arabia to bomb Yemen. They did not succeed, being stopped by security. Both admitted to their actions and their intent, arguing that they acted "for the greater good"*.
A district judge in Burnley accepted their beliefs were sincerely held and found them not guilty.
Roger Hallam sprayed the walls of Kings College, London, with the words "divest from oil and gas" in protest against its fossil fuel investments. He did not deny causing criminal damage estimated at £7,000 but argued that "his actions were lawful because there was an exemption in the Criminal Damage Act that permits damage if it protects others' property." While the judge ruled that the issue of climate change was "irrelevant" to the case, the jury still acquitted him of all charges.
Simon Bramwell, Ian Bray, Jane Augsburger, Senan Clifford, David Lamber, and James "Sid" Saunders were accused of causing criminal damage to Shell's London headquarters. They argued that their actions were a "necessary" and "proportionate" response to the harm caused by Shell.
Judge Gregory Perrins directed jurors that even if they thought the protesters were "morally justified", it did not provide them with a lawful excuse to commit criminal damage.
Despite this, the jury acquitted them.
Dr Diana Warner, Ruth Jarman, Ian Bray, Richard Barnard, Nick Cooper and Phil Kingston were charged with "obstructing trains or carriages on the railway" following their protest which targeted the Docklands Light Railway. They acknowledged their roles in the protest but "argued that their action was a lawful protest against government inaction on the climate crisis". They were found not guilty with a unanimous verdict.
I doubt this is an exhaustive list but I think it is sufficient to show that the arguments made by the defence in the trial of the Colston Four follow a well-established legal precedent and those arguments have been accepted by juries around the country. I don't know how many of these sorts of cases are unsuccessful, and it may be that successful defences are the exception rather than the rule. But the arguments made and the decision reached by the jury are not in any way unprecedented and anyone treating them otherwise needs to calm down.
* The greater good
it's okay to say "I don't know"
For an unsuccessful one, didn't them Heathrow Airport protestors a few years back get found guilty whilst giving a similar defence a go?
Fishnut wrote: ↑
The 'technically guilty but morally innocent' have been acquitted before. BoaF has already linked to these articles and I linked to this collection.
Exactly. Apart from them being Woodchopper links not BoaF. Juries can deliver perverse verdicts even when a judge directly orders them to ignore the defense argument. It's a fundamental aspect of the jury system that verdicts don't depend on the actual law.
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