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Non-invasive techniques to monitor the survival and migration of transplanted stem cells in real-time is crucial for the success of stem cell therapy. The aim of this study was to explore multimodality molecular imaging to monitor transplanted stem cells with a triple-fused reporter gene [TGF; herpes simplex virus type 1 thymidine kinase (HSV1-tk), enhanced green fluorescence protein (eGFP), and firefly luciferase (FLuc)] in acute myocardial infarction rat models.
Rat myocardial infarction was established by ligating the left anterior descending coronary artery. A recombinant adenovirus carrying TGF (Ad5-TGF) was constructed. After transfection with Ad5-TGF, 5×106 bone marrow mesenchymal stem cells (BMSCs) were transplanted into the anterior wall of the left ventricle (n = 14). Untransfected BMSCs were as controls (n = 8). MicroPET/CT, fluorescence and bioluminescence imaging were performed. Continuous images were obtained at day 2, 3 and 7 after transplantation with all three imaging modalities and additional images were performed with bioluminescence imaging until day 15 after transplantation.
High signals in the heart area were observed using microPET/CT, fluorescence and bioluminescence imaging of infarcted rats injected with Ad5-TGF-transfected BMSCs, whereas no signals were observed in controls. Semi-quantitative analysis showed the gradual decrease of signals in all three imaging modalities with time. Immunohistochemistry assays confirmed the location of the TGF protein expression was the same as the site of stem cell-specific marker expression, suggesting that TGF tracked the stem cells in situ.
Citation: Pei Z, Lan X, Cheng Z, Qin C, Xia X, Yuan H, et al. (2014) Multimodality Molecular Imaging to Monitor Transplanted Stem Cells for the Treatment of Ischemic Heart Disease. PLoS ONE 9(3): e90543. https://doi.org/10.1371/journal.pone.0090543
Editor: Christoph E. Hagemeyer, Baker IDI Heart and Diabetes Institute, Australia
Received: October 19, 2013; Accepted: January 31, 2014; Published: March 7, 2014
Copyright: © 2014 Pei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the National Natural Science Foundation of China (No. 30830041, 30571816, 30772208, and 30970853) (http://www.nsfc.gov.cn/Portal0/default152.htm), the China Postdoctoral Science Foundation (2005037194) (http://220.127.116.11/V1/Program1/Default.aspx), and Hubei Province Science Fund for Distinguished Young Scholars (2010CDA094) (http://www.hbstd.gov.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Although significant progress has been made in coronary revascularization and atherosclerosis prevention, cardiovascular diseases are still a major cause of death. Many animal and clinical experiments have demonstrated that treating ischemic heart disease with transplanted bone marrow mesenchymal stem cells (BMSCs) is feasible and promising [1]–[6]. Although traditional techniques such as in situ hybridization, PCR and immunohistochemistry are widely used to analyze the distribution and migration of transplanted stem cells, they are in vitro or post mortem and obviously not applicable for in vivo studies. Therefore, using non-invasive techniques to monitor the survival and migration of transplanted stem cells in real-time is crucial for the success of therapy.
In the past decade, techniques to monitor transplanted stem cells have reached a new stage in which the biological progress of transplanted tissues and cells can be monitored in vivo at the molecular level. A variety of cutting edge molecular imaging techniques have been developed [7], [8]. Different imaging methods have their advantages and disadvantages. Radionuclide imaging is highly sensitive but suffers low spatial resolution. Magnetic resonance imaging (MRI) shows the highest soft tissue contrast, but has low sensitivity. Bioluminescence and fluorescence imaging have relatively high sensitivity and spatial resolution, but cannot image deep tissues. To address these issues, multimodality molecular imaging has been actively developed in recent years. Many of these multimodality imaging techniques, such as radionuclide/MRI and optical imaging, are further used for cell trafficking such as stem cell monitoring [9]–[11]. These studies indicate that multimodality imaging has advantages over single-modality imaging. Furthermore, considering multimodality imaging probes and image fusion techniques have been developed rapidly, multimodality imaging may find many important applications in clinical practice.
The triple fusion gene TGF [herpes simplex virus type 1 thymidine kinase (HSV1-tk), enhanced green fluorescence protein (eGFP) and firefly luciferase (Fluc)] was recently developed and applied to stem cell monitoring [12], [13]. We have shown similar results in a previous study [14]. However, TGF has not been used to monitor transplanted stem cells in a myocardial infarction model. Therefore, in our current study, we aimed to determine whether TGF can be expressed in a myocardial infarction model using three imaging methods, and the duration of possible TGF expression. We explored the feasibility of multimodality imaging to monitor the transplanted stem cells in rat models with ischemic heart disease.
Materials and Methods
Ad5-TGF plasmid construction and recombinant adenovirus packaging
The pCDNA3.1 plasmid carrying the TGF fusion gene under the control of the CMV promoter was kindly provided by Dr. Sanjiv Sam Gambhir, Stanford University. The plasmid was repackaged into a recombinant adenovirus (Ad5-TGF) that was amplified and purified. The virus titer of 1.26×1010 TU/mL in 7 mL was determined by Vector Gene Technology Co., Ltd (Beijing, China).
Isolation and culture of BMSCs, and Ad5-TGF infection of BMSCs
All studies were approved by the Institutional Animal Care and Use Committee (IACUC) at Tongji Medical College, Huazhong University of Science and Technology (HUST). All the specific animals were maintained in the barrier system room at 22±2°C with an alternating 12 h light/dark cycle, and were given food and water ad libitum throughout the study period. All procedures in the animal experiment were carried out in strict compliance with the Guideline of Laboratory Animals Ethics Committee of Tongji Medical College, HUST. MSCs were isolated from the femur and tibia of a 4-week-old healthy Sprague-Dawley (SD) rat supplied by the Experimental Animal Center of Tongji Medical College, HUST (Wuhan, China). The bone marrow was flushed out with DMEM-F12 (Gibco, Langley, VA) containing 15% fetal bovine serum (FBS) (Gibco, Langley, VA), and then the cells were seeded in six-well plates. Passage 3–5 BMSCs cultured in OPTI-MEM serum-free medium (Gibco, Langley, VA) were infected with Ad5-TGF. The required volume of Ad5-TGF was calculated by the specific multiplicity of infection (MOI) of the BMSC count. Cells were slowly shaken inside a 37°C incubator for 2 h, and they were then transferred into a 37°C/5% CO2 incubator for 48 h.
Animal model
Thirty specific pathogen-free SD rats (200±25 g) were used to establish the animal model of acute myocardial infarction using previously published methods [15]–[18]. Rats were anesthetized with Isoflurane (Shanghai Abbott Laboratories, China) and fixed supinely on the operating table. The chest hair was shaved, and the skin was sterilized. A 2 cm incision was made at the left margin of the sternum in the third to fourth intercostal along the direction of the intercostal space. Deep and superficial fasciae were cut. The pectoralis major and serratus anterior muscles were separated to expose the ribs. After the bleeding stopped, the fourth intercostal muscle was separated, and the heart was quickly returned and the left anterior descending artery was ligated with a non-invasive suture. Each layer of the chest wall was sutured after the rat was able to breathe spontaneously.
BMSCs were infected with Ad5-TGF (MOI = 100) for 48 h. Rats (n = 22) that survived for 1 week after surgery were used for experiments. The method described above was used to extrude the heart. Ad5-TGF-infected BMSCs (5×106 cells in 100 µl) were slowly injected into the myocardium of the left ventricular anterior wall at the far end of the coronary artery ligation. Rats were transplanted with uninfected BMSCs for the negative control group (n = 8). In the experimental group (n = 14), five rats were performed microPET/CT and bioluminescence imaging continuously, and the other nine rats were used to obtain fluorescence imaging.
All rats for the model preparing were put on heat preservation pads during the surgery and till to fully awake. Surgical wound was prevented to expand during the surgery, which in turn minimized post-operative pain and distress. Penicillin (105 U/kg, every 24 hours) was intramuscularly injected for 4–5 days after the surgery in order to prevent infection. All rats were allocated to individual cage post-operation, and bred with nutritious pellets. After surgery, body weight, heart rate, respiration and specific behaviors of rats were recorded every 24 hours for at least one week. Rats were sacrificed after multimodality imaging with overdose anesthetic drug (pentobarbital, 150 mg/kg). All the animal experimental procedures were carried out under the guidance of the veterinaries in Department of Experimental Animals, Tongji Medical College, HUST.
Preparation of 18F-FHBG and microPET/CT imaging
A Siemens Inveon Acquisition Workplace (Siemens Preclinical Solution, Knoxville, TN) was used for microPET/CT imaging. After transplantation, rats were injected with 18F-FHBG (500±51 µCi) via the tail vein at days 2, 3 and 7, and prone position microPET/CT imaging was performed after 1 h for 20 min. The standard ordered-subset expectation maximization method was used for microPET image reconstruction. CT images were used for both attenuation correction of emission data and image fusion. Fusion of microPET/CT images showed horizontal, coronal and sagittal sections. Then, regions of interest (ROIs) were manually drawn. Quantitative analysis of 18F-FHBG uptake in the brain, heart, liver, lung, kidney, stomach, intestine and spleen was performed.
Bioluminescence imaging
A Maestro small animal imaging system (Cambridge Research & Instrumentation) was used for bioluminescence imaging. Rat models received an abdominal injection of 200 µl firefly luciferase substrate (10 mg/ml D-Luciferin; Xenogen, Alameda, CA). Bioluminescence imaging was performed for 5 min after injection. The size of the cooled charge coupled device (CCD) was 4×4 inches, and the operating temperature was –70°C. Bioluminescence imaging was performed at days 2, 3, 5, 7, 10 and 15 after transplantation.
Fluorescence imaging
A fluorescence imaging system for living animals (Roper Scientific, USA) was used. Rats (n = 3) were euthanatized at days 2, 3 and 7 after transplantation, respectively. The hair, muscle and ribs were removed to expose the thoracic cavity. The 488-nm excitation and 510-nm emission filters were used. The imaging time was 500 ms. Lumazone software was used for imaging and image analysis. Region of interests (ROIs) were manually drawn for quantitative analysis of the heart region. Three control rats were also used in fluorescence imaging.
PCR and immunohistochemistry
Total RNA was extracted from ex vivo myocardial tissue and reverse transcribed into cDNA. PCR primers for TGF were: forward, 5′-ATGCCCACGCTACTGCGG-3′; reverse: 5'-TCAGTTAGCCTCCCCCATCTC-3′. The PCR product size was 837 bp, and PCR conditions were 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 56°C for 30 s and 72°C for 1 min. The primers were provided by Invitrogen (Shanghai, China).
A portion of the ex vivo myocardial tissue was fixed with 4% formaldehyde, embedded in paraffin, and cut into 3 μm sections for hematoxylin and eosin (H&E) staining and immunohistochemistry analysis. An anti-HSV1-tk antibody (Santa Cruz, CA) was added to sections, followed by incubation at 4°C for 24 h. Then, a secondary antibody was added, followed by DAB staining, hematoxylin counterstaining, dehydration, and mounting. The stained sections were observed under an optical microscope and photographs were obtained.
The remaining ex vivo myocardial tissue was frozen in dry ice and cryosectioned into 15 μm sections. Sections were stained with anti-CD45 and anti-CD90 (eBioscience, San Diego, USA) antibodies at 4°C for 24 h, followed by incubation with a Cy3-labelled secondary antibody in the dark at room temperature for 1 h. Then, sections were counterstained with Hoechst 33258 to visualize cell nuclei, mounted with neutral glycerol, observed under a fluorescence microscope and photographed.
A serial of images of microPET, Fluorescence and Bioluminescence (Figure 1A) were obtained in acute myocardial infarction rats after transplanted Ad5-TGF-transfected BMSCs into myocardium at day 2, 3 and 7. Signals in the heart region could be clearly seen in different imaging modalities, whereas no signal could be found in the control group which transplanted with uninfected BMSCs (Figure 1A). Semi-Quantitative analysis results (Figure 1B, C and D) obtained by ROIs analysis of the heart region shows significant difference between the experimental group and the control group (P < 0.05) in all different imaging modalities.
A: From images of microPET (upper row), Fluorescence (middle row) and Bioluminescence (lower row), signals in the heart region could be clearly seen in different imaging modalities (indicated by red arrows) at day 2, 3 and 7 after transplantation of Ad5-TGF-transfected BMSCs into myocardium. Semi-Quantitative analysis results obtained by regions of interest (ROIs) analysis of the region of heart from 18F-FHBG microPET (B), Fluorescence (C) and Bioluminescence (D) imaging shows that significant difference could be seen between the experimental group with transplanted Ad5-TGF-BMSCs and the control group with transplanted uninfected BMSCs (P < 0.05) in all different imaging modalities.
MicroPET/CT imaging
MicroPET/CT was performed on modeled rats with transplanted BMSCs at 2 days after transplantation. The fusion image in Figure 2A shows obvious 18F-FHBG uptake in the myocardial infarction region of rats transplanted with Ad5-TGF-infected BMSCs. Next, quantitative analysis was performed. As shown in Figure 2B, 18F-FHBG uptake in the brain, heart, liver, lung, kidney, intestine, stomach and spleen of the modeled group was 0.016±0.005, 0.341±0.136, 0.413±0.179, 0.023±0.004, 0.472±0.104, 0.754±0.104, 0.653±0.164 and 0.531±0.112 percentage of injected dose per gram (%ID/g) (n = 5), respectively, while it showed 0.021±0.008, 0.011±0.014, 0.429±0.151, 0.028±0.009, 0.406±0.119, 0.772±0.107, 0.701±0.201 and 0.512±0.021%ID/g, respectively in the controlled group (n = 5). The heart/lung ratio of 18F-FHBG uptake of the modeled group was 31-fold higher than that of the negative control group (P = 0.043). Next, monitoring was performed for 1 week. As shown in Figure 1A and 1B, 18F-FHBG uptake in the heart region of the modeled group at days 3 and 7 after transplantation was 0.241±0.112 and 0.101±0.082%ID/g, respectively.
High signals (red arrow) could be clearly seen in the region of heart. B: Semi-quantitative analysis of 18F-FHBG uptake in different organs from the images. Significant difference exists in the heart between the experimental rats which were transplanted of Ad5-TGF transfected BMSC into myocardial infarction area and the control rats with non-transfected BMSCs only (n = 5, P < 0.05). However, no significant difference exists in other organs.
Bioluminescence imaging
Continuous monitoring of transfected stem cells was performed for 2 weeks by bioluminescence imaging of the same group of the myocardial infarcted rats that had already been scanned by microPET/CT (Figure 3A). Quantitative analysis at days 2, 3, 5, 7, 10 and 15 showed that the intensity of the bioluminescence signal in the heart region of rats in the modeled group was (3.556±0.725)×106, (2.731±0.652)×106, (1.946±0.531)×106, (0.962±0.326)×106, (0.662±0.266)×106 and (0.442±0.126)×106 photons/s/cm2/sr, respectively (Figure 3B) (n = 5). As a comparison, the intensity of the optical signal was only (0.033±0.03)×106 photons/s/cm2/sr in the heart region of rats in the negative control group (data not shown).
The red arrows indicate the signals in the heart region. B: Semi-quantitative analysis of images shows that the signal decreased with the time after transplantation (n = 5).
Fluorescence imaging
Continuous monitoring was also performed for 1 week by fluorescence imaging of transplanted BMSCs in myocardial infarcted rats. Fur, muscle and ribs were removed to expose the thoracic cavity (Figure 1A). Visible green fluorescence was detected in the heart region of rats in the modeled group, whereas no visible fluorescence was detected in the negative control group. Quantitative analysis showed that the fluorescence intensity in the heart region of rats in the modeled group at days 2, 3 and 7 was 582±107, 512±71 and 221±85 FU, respectively (n = 3). However, only 19±5 FU was measured in the heart region of rats in the negative control group (n = 3).
PCR and immunohistochemistry
After PCR and electrophoresis, specific DNA bands were detected between 750 and 1000 bp in DNA obtained from both the rat myocardial tissue and TGF-transfected BMSCs of the modeled group (Figure 4A). The DNA bands of cells were brighter than those of the tissue. No DNA bands were detected in the DNA from myocardial tissue of rats in the negative control group. H&E staining of myocardial tissue showed that Normal myocardial fibers displayed a regular and diffuse distribution (Figure 4B); however, infarcted myocardial fibers were swollen, disorganized, contained vacuoles and even broken (Figure 4C). Transplanted BMSCs distributed in the myocardial tissue gap were obvious, as shown in Figure 4D and E. eGFP fluorescence from transplanted BMSCs in the infarcted myocardium of rats in the modeled group was observed under a fluorescence microscope (Figure 4F). Positive staining (brown) of transplanted TGF-transfected BMSCs was detected by immunohistochemistry with an anti-HSV1-tk antibody and DAB staining (Figure 4G and H). No obvious staining was detected in normal myocardial cells. Immunofluorescence staining showed that transplanted BMSCs were positive for Cy3-CD90 (red, Figure 4I), but negative for Cy3-CD45 (Fig. 4K). Nuclei were stained and represented in blue (Figure 4J and 4L).
A: PCR result: 1. rat heart tissue transplanted with Ad5-TGF-transfected BMSCs; 2. Ad5-TGF-transfected BMSCs, 3. the control rat heart tissue with transplanted with non-transfected BMSCs; M, marker (from lower to upper: 100, 250, 500, 750, 1000 and 1500 bp). B: H&E staining of the heart tissue in healthy rats. C: H&E staining of the rat heart tissue with myocardial infarction; D and E: H&E staining of the rat heart tissue with BMSCs transplantation (BMSCs are indicated by black arrows, 100× and 400×, respectively); F: Frozen section of a rat in the modeled group with rBMSC transplantation (eGFP expression is indicated by red arrows); G and H: Immunohistochemistry of HSV1-tk in the heart tissue of rats in the modeled group with BMSC transplantation (brown indicates positive staining, 100× and 400×, respectively). I and K: Immunofluorescence of Cy3-CD90 and Cy3-CD45 in the heart tissue of rats in the modeled group with BMSC transplantation (CD90 is shown as red, CD45 was negative, 400×). J and L: Immunofluorescence of I and K with nuclear counterstaining (blue, 400×).
This study has shown that microPET/CT, fluorescence and bioluminescence imaging are non-invasive techniques that can be used to repeatedly monitor transplanted stem cells in animal models of myocardial infarction. We performed microPET/CT, fluorescence and bioluminescence imaging on each animal model of myocardial infarction at days 2, 3 and 7 after transplantation. Images of the transplanted region of the heart were even obtained by BLI at 15 days after transplantation. The semi-quantitative analyses of TGF expression obtained by the three imaging techniques were changing at the same trend over time. Finally, we verified the imaging results with the ex vivo assays using PCR and histological identification of the stem cell transplanted heart tissue. This study is the successful application of three different molecular imaging techniques to monitor transplanted stem cells in vivo in a myocardial infarction model.
Because stem cell transplantation is a valid treatment for ischemic heart disease, non-invasive molecular imaging methods have been actively pursued to monitor transplanted stem cells. First, PET reporter gene imaging is one of the most promising non-invasive molecular imaging tools, which is reliable and objective for locating transplanted stem cells in the myocardium of small animals and for quantitative analysis [12], [19], [20]. Willmann et al applied clinical PET to image large animals such as pigs, in which transplantation of human mesenchymal stem cells into the pig myocardium showed the feasibility of reporter gene imaging [21]. Subsequently, multimodality molecular imaging has been gradually developed and used to monitor transplanted stem cells in the myocardium. Higuchi et al monitored rat cardiac transplantation cell survival and positioning with both PET and MRI [11]. In a study by Wu et al [12], Fluc- and HSV1-sr39tk-transfected embryonic rat H9c2 cardiomyoblasts were transplanted into the myocardium of healthy mice, and in vivo monitoring was performed for 2 weeks using PET and BLI. However, these previous reports all used normal animals and are not an accurate reflection of stem cell survival in a lesioned environment. In this study, the major advantage is the success of continuous multimodality monitoring of stem cells in animal models of myocardial infarction, which is more intuitive and provides a reliable foundation for further applying biological therapy such as stem cells treatment in the future.
Using longitudinal monitoring with the three imaging techniques, we confirmed that BMSCs survived in lesions and did not migrate after transplantation. Based on quantitative analyses, we found that the signals in the heart region decreased as the monitoring time increased using the three imaging techniques. The signal intensity attenuated within 1 week, and by the second week the signal detected by microPET and fluorescence imaging was significantly reduced and could not be detected. Interestingly, BLI is highly sensitive, and in the second week the signal could only be detected by BLI. In our subsequent analyses, we evaluated the transplanted stem cells in the infarcted myocardium in which the local blood supply mechanism was significantly different from that in the normal myocardium. There may have been insufficient blood supply in the transplantation region, as well as the presence of lesions and inflammation, which could result in the death of some transplanted BMSCs in the infarcted region. The use of this infarction model is also the major difference compared with the normal rat study of Wu et al [12], which indicated that the survival of transplanted stem cells in the infarcted region was affected by the lesioned environment to a certain extent. One thing to note is that adenovirus was used as the TGF carrier, and it cannot insert the TGF fusion gene into the genome of BMSCs, resulting in the gradual reduction of exogenous proteins as a result of cell metabolism and proliferation.
We used the multi-functional reporter gene TGF for multimodality molecular imaging to monitor transplanted BMSCs for the treatment of ischemic heart disease. First, we combined microPET and CT technologies in which microPET provided functional imaging and CT provided accurate anatomical localization. As shown in Figure 2B, we precisely located the stem cell transplantation region by coronal, sagittal and cross sections. In the development of molecular imaging, regular conventional imaging has become an inseparable complement. We believe that in the future, PET/CT will be more applicable to clinical development of stem cell tracking techniques in vivo. Second, the sensitivity of BLI reaches a concentration of 10–15 mol, which is significantly superior to that of PET [22]. During the 2 weeks of monitoring in our study, PET and fluorescence imaging could only obtain images of the transplanted rats in the first week after cell transplantation, whereas BLI was able to monitor cells for the whole duration. However, the bioluminescence technique is limited in terms of the spatial resolution by the influence of light scattering, and the penetration of the optical signal is only 2 cm [23], [24], which is consistent with the images obtained in our study (Figures 1A and 3A). Thus, BLI has limited clinical use, and it is more suitable for small animal studies. Finally, owing to tissue attenuation and refraction, the eGFP of fluorescence imaging is only 2 mm [25], [26]. Because of interference by the fur and tissue of rats, thoracotomy is required before fluorescence imaging, as shown in Figure 1A. Fluorescence is an autonomous property of cells, and the generation of fluorescence does not require an exogenous reaction substrate. We directly analyzed the expression of eGFP in tissue under a fluorescence microscope. Moreover, fluorescence imaging is superior to the other two imaging techniques in terms of its use for the in vitro analysis of eGFP.
In our study, although the dynamic observation of survival and migration of stem cells in the myocardium of the infarction model was successful, the duration was relatively short for in vivo monitoring of stem cell proliferation and differentiation as well as evaluation of whether cardiac function improved after stem cell transplantation for treating ischemic heart disease. A recent study has shown that a retrovirus can insert a target gene into the genome of stem cells, which may be advantageous for monitoring stem cell proliferation [27]. Most current studies of in vivo monitoring of transplanted stem cells to treat ischemic heart disease have been focused on cell survival, proliferation and migration [20], [28]–[31]. Further research of stem cell differentiation and evaluation of its treatment efficacy is needed.
BMSCs promote myocardial repair and revascularization, and currently it is one of the promising methods for treating myocardial infarction [32]–[34]. To improve the repair of infarcted myocardium by transplanted BMSCs, a combination of gene therapy and transplanted BMSCs is used in most cases. For example, after transfection with Bcl-2 [35] or PAI-1 [36], the BMSC survival rate increases. Furthermore, Ang1-tranfected BMSCs provide better remodeling of infarcted myocardium [37]. Integrin-linked kinase promotes the adhesion of BMSCs to the infarcted myocardium [38]. Reporter gene imaging is mature and used for in vivo monitoring regardless of whether a therapeutic gene is expressed or not, the extent of expression and the duration of therapeutic gene expression [39]. In addition, owing to the characteristics the reporter gene technique, namely good specificity and a true reflection of the stem cells, such a technique is relatively mature for in vivo monitoring of stem cell therapy [40]. Therefore, TGF reporter gene imaging is likely to be a comprehensive method not only for tracking stem cells, but also for monitoring the gene expression in combination with gene therapy, which provides a multi-faceted platform for in vivo monitoring of transplanted stem cells for treating ischemic heart diseases.
This is the first application of TGF-transfected BMSC transplantation into the myocardial infarction model. Moreover, it proves that the dynamic situation of BMSCs in vivo can be monitored by microPET/CT, fluorescence and bioluminescence multimodality imaging. This study indicates that TGF can be used for in vivo monitoring of transplanted BMSCs for the treatment of ischemic heart disease as a multimodality reporter gene.
Author Contributions
Conceived and designed the experiments: XL YXZ. Performed the experiments: ZJP XL CXQ XTX HY ZLD. Analyzed the data: ZJP XL. Contributed reagents/materials/analysis tools: ZC ZJP. Wrote the paper: ZJP XL ZC YXZ.
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- 33. Schafer R, Northoff H (2008) Cardioprotection and cardiac regeneration by mesenchymal stem cells. . Panminerva Med. 50: 31–39.
- 34. Copland IB, Lord-Dufour S, Cuerquis J, Coutu DL, Annabi B, et al. (2009) Improved autograft survival of mesenchymal stromal cells by plasminogen activator inhibitor 1 inhibition. . Stem Cells. 27: 467–477.
- 35. Li W, Ma N, Ong LL, Nesselmann C, Klopsch C, et al. (2007) Bcl-2 engineered MSCs inhibited apoptosis and improved heart function. . Stem Cells. 25: 2118–2127.
- 36. Deuse T, Peter C, Fedak PW, Doyle T, Reichenspurner H, et al. (2009) Hepatocyte growth factor or vascular endothelial growth factor gene transfer maximizes mesenchymal stem cell-based myocardial salvage after acute myocardial infarction. . Circulation. 120: S247–254.
- 37. Sun L, Cui M, Wang Z, Feng X, Mao J, et al. (2007) Mesenchymal stem cells modified with angiopoietin-1 improve remodeling in a rat model of acute myocardial infarction. . Biochem Biophys Res Commun. 357: 779–784.
- 38. Song SW, Chang W, Song BW, Song H, Lim S, et al. (2009) Integrin-linked kinase is required in hypoxic mesenchymal stem cells for strengthening cell adhesion to ischemic myocardium. . Stem Cells. 27: 1358–1365.
- 39. Jaffer FA, Libby P, Weissleder R (2007) Molecular imaging of cardiovascular disease. . Circulation. 116: 1052–1061.
- 40. Higuchi T, Anton M, Saraste A, Dumler K, Pelisek J, et al. (2009) Reporter gene PET for monitoring survival of transplanted endothelial progenitor cells in the rat heart after pretreatment with VEGF and atorvastatin. . J Nucl Med. 50: 1881–1886.


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I have never set up a HAM radio station aboard. I would be interested in getting information on the antennas used on a sailboat.
Sue and Larry respond:
There are several options available to you. The majority of cruisers use their backstay as their antenna. This entails cutting your backstay at two locations, at least 23 feet apart, and fitting each location with an insulator designed for this purpose.
A second option is to use a 23-foot fiberglass whip antenna. Although this is a cheaper option than insulating your backstay, this often doesn't look very good on smaller boats. You may read about several other systems out there, but the above two are the most popular. You will also need proper grounding in your boat. This was covered in the sidebar of our "Choosing and Installing an SSB Radio" article. Good luck with your installation and get ready for some really interesting times.
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subset: commoncrawl


text:
Rohan Parkley and Soumya from Raghav Ke Rockstars gave beautiful performance in 9 June 2012 episode of ZEE TV Dance India Dance Lil Masters 2.
Rohan and Soumya performed a romantic dance for the song Suraj Hua Madham. They used Freestyle dancing style for the song.
Skipper Raghav Juyal loved Rohan and Soumya's dance. Master Geeta said that it was an innocent, sweet, cute dance act. Choreography is awesome, clean, nice and very well executed. She is proud to see Rohan and Soumya on the stage.
Master Marzi complimented that it very nicely incorporated lyrics with dance. Soumya is very cute and they are entrancing India with their dance.
Grand master Mithun da praised that Raghav did good choreography. Mithun da went on the stage and hugged Soumya. He gave grand salute and said Soumya that he is fallen in love with her.


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Venezuelan Winter League Notebook
News & notes for the week ending Nov. 25
November 27, 2007 5:00 AM ET
THE WEEK IN REVIEW: After losing five of six, the Aguilas went 3-2 last week and are 18-20.
WHO'S HOT: 3B Yurendell de Caster (Pirates) and 1B Randy Ruiz (Giants) combined for five homers, 19 RBIs and 12 runs scored over the past 10 games. ... SS Andres Blanco (Royals) hit .385 (5-for-13) and scored three runs. ... OF Rodney Medina went 8-for-12 (.667) with three runs scored and two RBIs in four games.
WHO'S NOT: OF Jonel Pacheco (Pirates) is hitless in his last 22 at-bats. ... OF Carlos Gonzalez (Diamondbacks) has eight hits in his last 36 at-bats (.222). ... 3B Pete Orr (Braves) is batting .154 (6-for-29) since Nov. 11.
NEWS AND NOTES: Ruiz and de Caster are tied for second in the league with six homers. ... P Rich Garces is second with 13 saves, while P Heath Totten (Phillies) is among the top 10 with four wins and a 2.75 ERA. ... Zulia is third in batting (.268) and runs scored (162), but leads the league with 249 strikeouts.
Bravos de Margarita
THE WEEK IN REVIEW: The Bravos have won seven straight to climb above .500 (20-19) for the first time this season.
WHO'S HOT: OF Frank Diaz (Nationals) is batting .500 (17-for-34) with two homers, nine runs scored and seven RBIs over an eight-game hitting streak. ... SS Alberto Gonzalez (Yankees) hit .436 (17-for-39) with a homer, seven RBIs and six runs scored over his last 10 games. ... P Jesus Silva (Marlins) is 6-1 with a 1.45 ERA in nine starts, limiting batters to a .225 average. He is first in the league in wins and second in ERA.
WHO'S NOT: 3B Julio Cordido (Giants) fell into a 4-for-22 (.182) slide. ... 1B Victor Rodriguez is 10-for-44 (.227) with a walk and six strikeouts since Nov. 13.
NEWS AND NOTES: Diaz and Rene Reyes are in the top 10 in batting. ... Alex Serrano (Angels) is tied for third with 10 saves. ... Margarita pitchers are second in ERA (2.87) and second in homers allowed (18) and are tied for the league lead with five shutouts.
Cardenales de Lara
THE WEEK IN REVIEW: The Cardenales split six games and are 21-16.
WHO'S HOT: Major League free agent SS Cesar Izturis has hit safely in nine of 11 games since joining Lara on Nov. 15 and has batted .435 (10-for-23) in his last six games. ... In his last 10 games, OF Charlton Jimerson (Mariners) is batting .289 (11-for-38) with a homer and six runs scored. ... P Brandon Morrow (Mariners) tossed a five-inning three-hitter in his debut on Nov. 20. ... P Cesar Jimenez (Mariners) extended his scoreless streak to 14 2/3 frames, while limiting batters to a .163 average.
WHO'S NOT: After a hot start, 2B Hernan Iribarren (Brewers) has two hits in his last 17 at-bats (.117). ... P Yovany D'Amico (Royals) has allowed runs in five straight appearances with an 8.25 ERA over that span.
NEWS AND NOTES: OF Robert Perez is tied for the league lead with 28 RBIs. ... Jimerson is third in the league with six stolen bases. ... Lara leads the league in homers (27), but is seventh in batting (.250).
Caribes de Anzoategui
THE WEEK IN REVIEW: The first-place Caribes won two of seven games last week and fell to 24-16. They lead Lara by 1 1/2 games.
WHO'S HOT: OF Jody Gerut hit .375 (9-for-24) in seven games and continues to lead the league with a .390 batting average. He also ranks first in hits (55), on-base percentage (.488) and runs scored (34), third in slugging percentage (.567) and second in triples (3). ... IF Omar Infante (Cubs) hit .308 (8-for-26) with two homers, six runs scored and five RBIs in seven games and ranks second with 28 runs scored. ... IF Rico Washington (Cardinals) leads the league with 28 RBIs ... OF Andres Torres (Tigers) ranks second in stolen bases (7), second in runs scored (28) and fifth in slugging percentage (.551).
WHO'S NOT: OF Gorkys Hernandez (Braves) went 1-for-8 with four strikeouts in five games, dropping his batting average to .152. ... C Eliezer Alfonzo (Giants) hit .087 (2-for-23) in seven games and is batting .213 in 34 games.
NEWS AND NOTES: Mike Smith (Cardinals) won his third straight start on Nov. 24, tossing seven shutout innings in a 4-1 win over Caracas. The 30-year-old right-hander hasn't yielded an earned run or a walk and has struck out 20 over his last 21 innings. ... The Caribes are fourth in the league with a .264 average and a 3.67 ERA.
Leones del Caracas
THE WEEK IN REVIEW: The Leones won four of six games but sit in last place at 15-22.
WHO'S HOT: C Carlos Maldonado hit .458 (11-for-24) with four RBIs in six games and is batting .512 (22-for-43) over an 11-game hitting streak. ... OF Drew Macias (Padres) hit .385 (5-for-13) with four runs scored in his first four games. ... IF Jose Castillo (Pirates) is fourth in the league with a .346 batting average and ranks second with a .579 slugging percentage. ... IF Mario Lisson (Royals) leads the league with seven homers and OF Carl Loadenthal (Braves) has a league-best 11 stolen bases. ... Athletics farmhand Brad Knox (1-1, 3.35) ranks seventh in ERA.
WHO'S NOT: IF Omar Vizquel (Giants) hit .150 (3-for-20) in six games and is hitting .170 in 14 contests. ... OF Roger Cedeno has two hits in his last 31 at-bats and is hitting .158 in 22 games.
NEWS AND NOTES: LHP Eric Cyr (Dodgers) pitched seven shutout innings in a 4-1 win over Aragua on Nov. 20. ... RHP Carlos Bohorquez (Reds) picked up victories in consecutive relief appearances. ... The Leones rank sixth in hitting with a .251 average and fifth in pitching with a 4.07 ERA.
Navegantes del Magallanes
THE WEEK IN REVIEW: The Navegantes went 2-4 last week and have lost three straight to fall into sixth place at 16-19.
WHO'S HOT: OF Richard Hidalgo, a former Major Leaguer, is batting .414 (24-for-58) with six homers, 13 RBIs and 13 runs scored over a league-best 15-game hitting streak. ... 2B Tomas Perez (White Sox) batted .321 (17-for-53) during a 14-game hitting streak that was snapped on Nov. 25. ... OF Juan Rivera (Angels) is batting .450 (9-for-20) in his last five games.
WHO'S NOT: In their last 10 games, Astros prospects Mark Saccomanno and Luke Scott combined for seven hits in 54 at-bats (.130).
NEWS AND NOTES: Hidalgo (.352) and Perez (.339) are third and fifth in hitting. ... The Navegantes' offense is second in batting (.268) and walks (147), but the pitching staff is seventh in ERA (4.13) and has allowed a league-worst 29 homers.
Tiburones de La Guaira
THE WEEK IN REVIEW: The Tiburones went 2-4 and are in seventh place with a 16-20 record, six games off the pace.
WHO'S HOT: OF Gregor Blanco (Braves) hit .300 (6-for-20) with a pair of homers and four RBIs in six games. He ranks fifth in the league with a .418 on-base percentage and fourth with 26 runs scored. ... IF Chris Carter (Red Sox) is second with a .355 batting average and 49 hits and fourth with 24 RBIs. ... LHP Stephen Randolph fanned eight over six shutout innings in a 3-2 win over Lara on Nov. 24. ... RHP Jeff Bennett (3-1, 1.25) ranks first in ERA and has won three straight decisions.
WHO'S NOT: OF Ron Acuna went 3-for-18 with four strikeouts in six games and is hitting .233 in 35 games. ... OF Jackson Melian is batting .071 (1-for-14) in his last 10 games and is hitting .100 (3-for-30) with eight strikeouts in 15 contests.
NEWS AND NOTES: RHP Enrique Gonzalez (Nationals) yielded two earned runs while fanning eight in 14 innings over three starts. ... The Tiburones are eighth in the league with a .239 average and rank fifth in pitching with a 3.77 ERA.
Tigres de Aragua
THE WEEK IN REVIEW: The Tigres are in third place with a 20-18 record after losing three of five games last week.
WHO'S HOT: C Mike Rivera (Brewers) hit .385 (5-for-13) with two RBIs in his first four games. ... IF Luis Rodriguez (Padres) led the league with seven runs scored in five games ... IF Ronny Cedeno (Cubs) hit .368 (7-for-19) in five games and ranks eighth with a .325 average. He also ranks third in doubles (10) and fourth in on-base percentage (.420). ... RHP Cory Bailey (Cubs) is second with 31 strikeouts.
WHO'S NOT: IF Mike Aviles went 2-for-9 in two games and is hitting .194 (7-for-36) with 10 strikeouts in his last 10 games. ... Twins prospect Brian Bass (3-3, 3.38) has lost two straight starts but still ranks eighth in ERA and fourth with 29 strikeouts.
NEWS AND NOTES: LHP Horacio Estrada (2-1, 1.80) tossed 5 1/3 shutout frames in a 1-0 win over the first-place Caribes on Nov. 21 and has yielded one earned run in 14 1/3 frames over his last three starts. The 32-year-old ranks third in the league in ERA. ... The Tigres rank fifth in hitting with a .258 average and their pitchers lead the league with a 2.85 ERA.
Steve Conley and Shane Figueroa are contributors to MLB.com. This story was not subject to the approval of the National Association of Professional Baseball Leagues or its clubs.View More


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Batman Dark Flight
Prepare yourself for a pulse-racing adventure on DC Comics' first Batman 4D flight simulation ride.
Enhanced with the latest in flight simulation technology and the very best in audio design and visual graphics, "Batman Dark Flight" takes you, and the legend of Batman, to a whole new dimension of super-heroic entertainment. This incredible multi-sensory, action-packed ride throws you among all of Batman's heart stopping encounters as he combats the hair-raising terror of Gotham City's Super-Villains.
As an integral part of the storyline, you will join Batman to battle through the explosive action and heart-stopping gunfire on the streets of Gotham City - but this will be no easy task as it is notorious criminals like The Joker, Two-Face and Bane who are out to inflict damage and destruction on the mission.
From flying through the city in the Batplane to racing around the streets in a high-speed car chase, this pulse-racing adventure is filled with the risk of disaster - and it's up to you and Batman to save Gotham City from the devastation.
Limited time offer: Present your Studio City hotel keycard or The House of Dancing Water ticket to purchase a Batman Dark Flight ticket at a discounted price of MOP100*
*Offer is valid from the day of check-in until the day of check-out and on the show date and the date after The House of Dancing Water show. Other terms and conditions apply.
During Macau's typhoon season (May to September), the operating hours of our entertainment attractions (including "Golden Reel", "Batman Dark Flight", "The House of Magic" and "Warner Bros. Fun Zone") might be affected by inclement weather. Please see below for the attractions' operating hours under the respective weather warnings:
Thunderstorm Warning
- "Golden Reel" will be suspended due to poor weather condition. It will resume to normal operation approximately 1 hour after the warning is lowered.
- "Batman Dark Flight", "The House of Magic" and "Warner Bros. Fun Zone" remain open.
Typhoon Signal No. 3
- "Golden Reel" will be suspended due to poor weather condition. It will resume to normal operation approximately 3 hours after the signal is lowered.
- "Batman Dark Flight", "The House of Magic" and "Warner Bros. Fun Zone" remain open.
Typhoon Signal No. 8 or above
- "Golden Reel" will be suspended due to poor weather condition.
- "Batman Dark Flight", "The House of Magic" and "Warner Bros. Fun Zone" may suspend operation due to poor weather condition.
- Guest must be between 100 cm and 195 cm to ride this attraction.
- Guests between 100 cm and 140 cm must be accompanied by an adult over the age of 18.
- Guests weighing more than 136kg are not permitted to ride this attraction.
- Any disabled guests must be able to move from the load position into the seat at their own accord or with their own assistance within their party.
- Guests with the following conditions are prohibited from experiencing this attraction:
- Expectant Mothers
- Heart conditions or high blood pressure
- Back or neck pain
- Motion sickness, dizziness or epilepsy
- Sensitivity to flashing flights
- Claustrophobia
- In order to enjoy the best experience and remain safe on the attraction please:
- Remain seated at all times
- Ensure that your safety harness is fastened and secure
- Secure all loose articles before loading
- Ensure that your belongings are stored in the storage compartment provided
- Keep your hands free at all times and inside the ride
- The following are not permitted on this attraction:
- Food or drink
- Photography or video recording
- Smoking
- In the event of an emergency, our evacuation procedures will be activated, ensuring that you are kept safe and unharmed during the event
- The management reserves the right to refuse entry of any guest who are deemed unfit to enter.
- For safety reasons, you may be requested to show your bags and belongings for inspection upon entry.
Any person or entity purchasing tickets to Attractions, Shows, or Events agrees to be bound by these Terms &Conditions. Studio CityHotels Limited and Attractions (the "Management") reserve the right to amend, delete, and/or add to these Terms &Conditions without prior notice at its sole and absolute discretion. The Macau Law shall govern these T&C and all purchasers of tickets and/or Guests agree to submit to the exclusive jurisdiction of the Macau courts.
- The ticket is purchased for the show and session as specified on the tickets but the date and time of the show may be subject to change. In such instance, there is no obligation to refund or exchange the tickets. All tickets sold are subject to Management's right to cancel, alter and/or reschedule the performance, add, withdraw or substitute artists, vary advertised programs, change the prices, venues or audience capacity.
- All ticket sales are final. All completed and confirmed transactions cannot be cancelled, and are non-refundable and non-exchangeable for any reason. Management is not liable for any lost, stolen or destroyed tickets.
- Tickets should only be purchased directly from Management authorized sales channels and official ticket agents. Any person holding a ticket that is acquired through a non-authorized sales channel such as resold tickets will be refused admission to the show or attraction.
- Tickets may only be used by the holder to gain admission to the relevant show or attraction and may not be resold or offered for resale at any price in excess of the initial purchase price (whether through online auction sites or otherwise) or be used for or in connection with advertising, promotion or other commercial purposes (including without limitation to competitions and trade promotions) or to enhance the demand for other goods or services, either by the original buyer or any subsequent bearer, unless authorized by Management. Management reserves the right to cancel any tickets that have been resold and to deny any such Guest entry to the show or attraction without a refund.
- Management shall not be liable for any complaints, claims, refunds, or exchange for any reason and shall not be deemed to be in breach of these Terms &Conditions. Neither is Management liable for any delay or non-performance which is due to any cause beyond its reasonable control, or for any costs incurred other than the cost of the tickets.
- One person per ticket regardless of age. Persons purchasing, claiming or collecting purchased tickets are required to present their valid photo ID, confirmation number, the credit card used for purchase and/or relevant confirmation documents (if applicable).
- Tickets are non-transferable, non-exchangeable, non-refundable, not for re-sale and are revocable. Tickets are void if altered.
- Paper and electronic tickets are printed with unique reference numbers and security features. Holders of any other ticket type will be refused admission to the show or attraction and may face prosecution.
- Management reserves the right to cancel or modify redemptions where it appears that a ticket holder has engaged in fraudulent or inappropriate activity or under other circumstances where it appears that the redemptions contained are the result of a mistake or error.
- Travel to and from the venue is the sole responsibility of the Guest. No refund and exchange will be permitted due to the alteration, late arrival, unavailability, postponement or cancellation of travel services by land, air or sea.
- Guests attending the show or riding attraction do so at their own risk. The Guest voluntarily assumes all risks and danger incidentals to the show or attraction whether occurring before, during or after the show or riding attraction. Management is not responsible for any loss, damage, personal injury, liability or death so caused. The Guest assumes all risks and liabilities for any medical conditions associated with attending the show or riding attraction. Management shall bear no liability for any personal injuries, health problems or death sustained at a show or in attraction, whether it is directly or indirectly resulting from any existing medical condition of the Guest.
- Management reserves the right to alter the seat allocation by providing alternative seats to those specified on the ticket without prior notice. If this is necessary for any reason, Management will reimburse any reduction in value between the original and altered seat allocation.
- Any unauthorized soliciting, selling of goods, tickets, or services or guest surveys of any kind are prohibited.
- Management reserves the right to refuse admission to any person whose conduct is disorderly, discourteous,or inappropriate or who poses a threat to security or to the enjoyment of the show without refund or compensation. Customers who cause a disturbance, act inappropriately or refuse to comply with requests made by Management will be evicted from the attractions and theaters without any refund or compensation.
- For security purposes and upon request, the ticket holder may be subject to inspection and for these purposes must open any bag, package or other item which is in his or her possession before admission.
- Any item (includes but not limit to glass bottles, cans, or knives) or restricted object deemed by the Management to be dangerous or inappropriate is not permitted for admission.
- All mobile phones, portable communication devices or any other electronic equipment should be disabled when inside the venue to avoid disturbing the performance.
- Guests must be appropriately clothed and must wear shoes at all time. Guests not appropriately clothed and/or with bare feet will not be permitted for admission.
- Management will not be liable for any delay or cancellation due to any force majeure such as power failure, fire, explosion, flood, typhoon, storm or similar disasters, strikes, industrial disputes, abnormally inclement weather, war, insurrection, riot, civil disturbance, acts or threats of terrorism, act of God, communicable disease outbreaks, industrial action, acts or regulations of national or local governments, governmental order or decrees, or any other cause beyond its reasonable control.
- Without prejudice to clause 5 & 19, in the event of cancellation or postponement, Management will only be responsible for ticket exchange or any full / partial refund of the ticket, whether purchased as part of a package or not. Any refund shall be made to the holders of the original tickets only.Complimentary tickets are not eligible for cash refunds.
- Tickets are issued/sold on behalf of the organizer/presenter of the event whose name appears on the ticket. For any event cancellation or postponement, the organizer/presenter shall be responsible for refunding the purchase price of the tickets and the refund shall be made to the holders of original tickets only.
- In the event of disputes, Management reserves the right to make final judgment on the dispute and otherwise in respect of tickets and admissions.
- As per local law, smoking is prohibited in all Studio City attractions and theaters.
- Guests must abide by and follow all public announcements, directions, restrictions, warnings, or regulations in relation to the entry to and remaining within all Studio City venues.
- Complimentary tickets are not eligible for cash refunds. In the event of closure, delay or postponement, holders of complimentary tickets should contact the department that offered the ticket for ticket exchange.
- For restrictions regarding:height, pregnant/Expectant Mothers, Health Conditions, Latecomers, Parental Consent, Age, and Photo & Video policies please refer to Venue Conditions of Entry.
- Performances may contain flashing lights, loud audio effects, fog, pyrotechnics, hydro effects, animatronic, lighting and other special effects. Management will not be held liable for any health issues directly resulting from attending the show at the attractions and theaters.
- In the event of any conflict or inconsistency between the English version and the Chinese version of these Terms and Conditions, the English version shall prevail.
- Management reserves the right of final decision on the amendments and interpretation of these terms and conditions without prior notice.
11am - 9pm (Sat, Sun & Public Holidays)
Please arrive 30 minutes before the attraction closing time
Please visit "THE HOUSE OF MAGIC" Box Office on Level 2, Studio City.
Opening Hours: 11am to 10pm (Mon – Sun).


text:
The 64-year-old president of Diagnostic Outpatient Centers, Williams also ran for mayor in 2001.
Larry Williams hopes people remember what made him popular almost a decade ago. His work in the neighborhoods of south St. Petersburg. His quest to make education a part of the city government's portfolio. His ability to mediate often tense and bitter council exchanges. But when it comes to running for mayor, Williams is hoping for different results. ... Read more
Larry Williams raised $100 from one donation in the first quarter. Totals do not include candidates' loans or donations to their own campaign.
*Totals do not include candidates' loans or donations to their own campaign.
Get profiles, links, campaign finance reports and more:
The issues are as varied as the opinions. See what
the candidates think about:
The Timessurveyed 600 registered St. Petersburg voters between June 11-16.
More than $250,000 has been raised by
candidates so far, setting the stage to make this the most expensive
mayoral race in city history. See where the money's coming from.
More coverage from the St. Petersburg Times and tampabay.com:
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text:
LONDON • Britain's Vodafone PLC, one of the world's largest cellphone companies, confirmed Thursday that it was talking to Verizon Communications about selling its stake in Verizon Wireless, the No. 1 mobile carrier in the U.S.
The U.K. company is mulling its options for its 45 percent stake in Verizon Wireless. Verizon Communications owns the other 55 percent.
Analysts have suggested that Verizon wants to pay around $100 billion for Vodafone's stake, although reports have said that U.K. group is pressing for as much as $130 billion.
Verizon Communications' stock rose $1.26, or 3 percent, to close at $47.82 on Wednesday. It had jumped $3.55, or 7.6 percent, to $50.11 in premarket trading about an hour before the stock market opened. Meanwhile Vodafone's London-listed share price rose 8.8 percent to 2.06 pounds.


text:
All, I've search but no pictures - only chat about it. As part of preventive maintenance, I'd like to clean the Throttle-Body on the R car - 2005. Does anyone know how to get to it from on top of the car - DIY step-by-step and/or pictures of where the dang thing is within the engine bay?
OCEAN stated that "the easiest way to clean it was to loosen the intake hose clamp on the throttle and move the pipe to the side. Then clean the throttle from underneath the car. Using a long screwdriver to keep the throttle opened, spray carb cleaner up in there. Its a lot easier than removing the throttle. The butterfly and the bore can get all gunked up. A good cleaning isn't a bad idea at 60K."
I'd rather remove it and do it myself. Anyone know how?


text:
Judge orders prosecution to prove that Bradley Manning intended to 'aid the enemy'
A US military judge says the government must prove that Army Private Bradley Manning intended to aid the enemy when he released hundreds of thousands of sensitive files to WikiLeaks.
The official tribunal against the 25-year-old private first class is not slated to start until June, but during pretrial hearings on Wednesday morning, Col. Denise Lind ruled that the government has a burden to prove that the soldier aimed to inflect harm on the United States when he sent materials to the whistleblower site.
Reporting from the media center at Ft. Meade, Maryland, independent journalist Alexa O'Brien writes that the United States government must now prove that Pfc. Manning acted "with reason to believe such info could be used to the injury of the US or to advantage of any foreign nation." By doing so, Lind is making it much harder for the government to convict Manning on the most serious of the charges: aiding the enemy. If convicted on this count, Manning could theoretically be sentenced to die. Prosecutors, however, have said they would settle for life in prison.
Joshua Michael Hakken And Family Will Be Turned Over To US Officials By Cuban Authorities
HAVANA — Cuba says it will turn over to the United States a Florida couple who allegedly kidnapped their own children from the mother's parents and fled by boat to Havana.
Foreign Ministry official Johana Tablada told The Associated Press in a written statement Tuesday that Cuba had informed U.S. authorities of the country's decision to turn over Joshua Michael Hakken, his wife Sharyn and their two young boys. She did not say when the handover would occur.
North Korean attack on Austin would be Obama's wet dream
April 6, 2013 by supermario
Filed under Commentary
As the wacky and almost comical war rhetoric between North Korea and the United States escalates, the U.S. corporate media outlets have recently reported that Kim Jong Un is making preparations to 'hit U.S. mainland targets.' One of those alleged targets is Austin, Texas… out of all places. Setting aside the fact that North Korea has no capabilities to hit the U.S. mainland with missiles, or at least the lower 48 states, let's examine why Kim Jong Un would choose Austin, Texas.
Let's suppose that North Korea, after arduous research, testing, and scientific breakthroughs, finally develops a missile that can reach the U.S. mainland. Let's suppose that Kim Jong Un, the 'dear leader' of North Korea has an option to hit a variety of targets in the U.S., like the San Diego Navy Base and Camp Pendleton on the west coast, or perhaps Fort Benning, Naval Station Norfolk, or any other military location that can really cripple the abilities of the United States to launch a counter-attack.
Why in the world would North Korea choose Austin, Texas? And if dear leader Kim Jong Un hates Texas so much, then why not choose other targets in Texas? Why not San Antonio, which has three huge air force and army bases? Why not Fort Hood, which on a daily basis prepares thousands of army troops for overseas deployments? Why not Fort Bliss, Naval Air Station Corpus Christi or NAS Kingsville?
Austin would be of very little military value to a foreign enemy that would be crazy enough to hit the U.S. mainland. The only military base there is Camp Mabry, home of the Texas Military Forces – the Texas National Guard and the Texas State Guard. If Texas were to secede from the U.S., this would be the central location for all military operations of a newly independent state.
Uh-oh! Did I just mention the forbidden word 'secession'? Shhhhhh….!
Austin is also the capital of Texas, which wants back its $1 Billion worth of gold from the federal government. Texas is also one of the leading states opposing Obamacare and Obama's gun control agenda. Therefore, wouldn't you say that if Kim Jong Un's alleged war plans were real, and they were not photoshopped by the New York Times as many allege, wouldn't he be doing Obama a favor by attacking Austin?
Nonetheless, this is just a hypothetical scenario; a North Korean attack on Austin would probably never happen. But if it did, and North Korea doesn't have the capabilities to carry it out, then who would really be behind it? And wouldn't this be the perfect pretext to go to war against North Korea and eliminating domestic political opponents at the same time?
Obama to let U.S. spy agencies scour Americans' finances
March 14, 2013 by Jack Blood
Filed under Police State
NEW YORK/WASHINGTON (Reuters) – The Obama administration is drawing up plans to give all U.S. spy agencies full access to a massive database that contains financial data on American citizens and others who bank in the country, according to a Treasury Department document seen by Reuters.
The proposed plan represents a major step by U.S. intelligence agencies to spot and track down terrorist networks and crime syndicates by bringing together financial databanks, criminal records and military intelligence. The plan, which legal experts say is permissible under U.S. law, is nonetheless likely to trigger intense criticism from privacy advocates.
Financial institutions that operate in the United States are required by law to file reports of "suspicious customer activity," such as large money transfers or unusually structured bank accounts, to Treasury's Financial Crimes Enforcement Network (FinCEN).
The Federal Bureau of Investigation already has full access to the database. However, intelligence agencies, such as the Central Intelligence Agency and the National Security Agency, currently have to make case-by-case requests for information to FinCEN.
The Treasury plan would give spy agencies the ability to analyze more raw financial data than they have ever had before, helping them look for patterns that could reveal attack plots or criminal schemes.
The planning document, dated March 4, shows that the proposal is still in its early stages of development, and it is not known when implementation might begin.
Financial institutions file more than 15 million "suspicious activity reports" every year, according to Treasury. Banks, for instance, are required to report all personal cash transactions exceeding $10,000, as well as suspected incidents of money laundering, loan fraud, computer hacking or counterfeiting.
"For these reports to be of value in detecting money laundering, they must be accessible to law enforcement, counter-terrorism agencies, financial regulators, and the intelligence community," said the Treasury planning document.
A Treasury spokesperson said U.S. law permits FinCEN to share information with intelligence agencies to help detect and thwart threats to national security, provided they adhere to safeguards outlined in the Bank Secrecy Act. "Law enforcement and intelligence community members with access to this information are bound by these safeguards," the spokesperson said in a statement.
Some privacy watchdogs expressed concern about the plan when Reuters outlined it to them.
A move like the FinCEN proposal "raises concerns as to whether people could find their information in a file as a potential terrorist suspect without having the appropriate predicate for that and find themselves potentially falsely accused," said Sharon Bradford Franklin, senior counsel for the Rule of Law Program at the Constitution Project, a non-profit watchdog group.
Despite these concerns, legal experts emphasize that this sharing of data is permissible under U.S. law. Specifically, banks' suspicious activity reporting requirements are dictated by a combination of the Bank Secrecy Act and the USA PATRIOT Act, which offer some privacy safeguards.
National security experts also maintain that a robust system for sharing criminal, financial and intelligence data among agencies will improve their ability to identify those who plan attacks on the United States.
"It's a war on money, war on corruption, on politically exposed persons, anti-money laundering, organized crime," said Amit Kumar, who advised the United Nations on Taliban sanctions and is a fellow at the Democratic think tank Center for National Policy.
The Treasury document outlines a proposal to link the FinCEN database with a computer network used by U.S. defense and law enforcement agencies to share classified information called the Joint Worldwide Intelligence Communications System.
The plan calls for the Office of the Director of National Intelligence – set up after 9/11 to foster greater collaboration among intelligence agencies – to work with Treasury. The Director of National Intelligence declined to comment.
More than 25,000 financial firms – including banks, securities dealers, casinos, and money and wire transfer agencies – routinely file "suspicious activity reports" to FinCEN. The requirements for filing are so strict that banks often over-report, so they cannot be accused of failing to disclose activity that later proves questionable. This over-reporting raises the possibility that the financial details of ordinary citizens could wind up in the hands of spy agencies.
Stephen Vladeck, a professor at American University's Washington College of Law, said privacy advocates have already been pushing back against the increased data-sharing activities between government agencies that followed the September 11 attacks.
"One of the real pushes from the civil liberties community has been to move away from collection restrictions on the front end and put more limits on what the government can do once it has the information," he said.
Michigan Senate Unanimously Passes NDAA Nullification Bill
March 13, 2013 by Kristen
Filed under Police State
By a vote of 37-0, the Michigan state Senate approved a bill preventing the president from executing the indefinite detention provisions of the National Defense Authorization Act (NDAA) inside the sovereign borders of the Wolverine State.
Senate Bill 94 — a companion measure to House Bill 4138 passed unanimously by the state House of Representatives last year — prohibits "any agency of this state, any political subdivision of this state, any employee of any agency of this state or any political subdivision of this state, or any member of the Michigan national guard from assisting an agency of the armed forces of the United States in the investigation, prosecution, or detainment of any citizen of the United States" under certain situations.
Particularly, the bill applies to any attempt by a state agent or officer to enforce "section 1021 of the national defense authorization act for fiscal year 2012, if such aid would place that state agency, political subdivision, employee, or member of the Michigan national guard in violation of the United States constitution, the state constitution of 1963, or any law of this state."
Wary of Attack With Smallpox, U.S. Buys Up a Costly Drug
The United States government is buying enough of a new smallpox medicine to treat two million people in the event of a bioterrorism attack, and took delivery of the first shipment of it last week. But the purchase has set off a debate about the lucrative contract, with some experts saying the government is buying too much of the drug at too high a price.
A small company, Siga Technologies, developed the drug in recent years. Whether the $463 million order is a boondoggle or a bargain depends on which expert is talking. The deal will transform the finances of Siga, which is controlled by Ronald O. Perelman, a billionaire financier, philanthropist and takeover specialist.
Smallpox was eradicated by 1980, and the only known remaining virus is in government laboratories in the United States and Russia. But there have long been rumors of renegade stocks that could be sprayed in airports or sports stadiums. Experts say the virus could also be re-engineered into existence in a sophisticated genetics lab.
California seizes guns from owners – and it might become a national model
March 12, 2013 by Kristen
Filed under Police State
In California, the government is already coming for the guns.
Notwithstanding the Second Amendment, rules and regulations across the United States outline certain restrictions for who can legally possess a firearm. In the state of California, factors such as a felony conviction or a history of mental health issues mean roughly 20,000 gun owners are holding onto their firearms illegally. Slowly but surely, though, Golden State police officers are prying them away. There's more, though: backers of the program suggest this becomes a nation-wide practice, and are asking the White House to help make it happen.
"Very, very few states have an archive of firearm owners like we have," Garen Wintemute of the Violence Prevention Research Program tells Bloomberg News. Wintemute helped set up a program on the West Coast that monitors not just licensed gun owners but also watches for any red flags that could be raised after admittance to a mental health institute or a quick stint in the slammer.
Wintemute says that as many as 200,000 people across the United States may no longer be qualified to own firearms, and in California they are making sure that number drops day by day. In one example cited in this week's Bloomberg report, journalists recall a recent scene where nine California Justice Department agents equipped with 40-caliber Glock pistols and outfitted in bulletproof vests knocked on a suburban residence, requested to speak to a certain gun owner and then walked away with whatever arsenal they could apprehend.
U.S., South Korea begin military exercises as North ends armistice
(CNN) — A new joint military exercise between South Korea and the United States began Monday amid heightened tensions across the region.
In a sign of crumbling relations, North Korea refused to answer its hotline with Seoul, South Korea's unification ministry said Monday, according to the Yonhap news agency.
The ministry said the North did not answer two attempts to communicate by telephone at 9 a.m. and 4 p.m. local time.
The latest military drills involving the two allies are called Key Resolve. They follow the Foal Eagle joint exercises that began March 1, which are scheduled to last two months. More than 3,000 U.S. forces are taking part in Key Resolve, according to United States Forces Korea.
The latest exercises fall under the shadow of North Korea's army declaring invalid the armistice agreement that ended the Korean War in 1953, an article in Rodong Sinmun, the official newspaper of North Korea's ruling Workers' Party, reported Monday.
WAR ON AFGHANISTAN; Karzai Accuses US & Taliban Of Working Together To Coordinate Bombings During Chuck Hagel's Visit
"Are the US government and the Afghan Taliban working together? Afghan President Hamid Karzai seems to think so after a couple of bombings in Kabul yesterday. He said they were carried out by the Taliban in coordination with the American government. The United States is supposed to withdraw most troops by the end of next year, and Karzai suggested the US is using terror attacks to preview how things will turn, once they leave." – FOX News Radio
I just happened to catch this report by FOX News Radio, only once, and after searching YouTube for more details I realized just how poorly reported this very important story is. See audio file below of the FOX News Radio report which I saved. The only YouTube video with any details follows this. I've also included another video from last December when Karzai accused the US, NATO, and "Private Security Firms" of destabilizing the country, saying, "there is a very strong perception that some of that insecurity is intentional."
2013.3.10 Are The US Government & Afghan Taliban Working Together? (FOX News Radio):
2013.3.10 Afghan Accusations; Karzai Says US & Taliban Are Cooperating To Worsen Security Situation (JewishNewsOne, youtube.com):
Afghan President Hamid Karzai has accused the Taliban and the US of working together to convince Afghans that violence will worsen if foreign troops leave the country. Karzai said that two deadly suicide bombings over the weekend demonstrated that the insurgent group is conducting attacks at the service of America. He said both Washington and the Taliban want US forces to stay beyond the end of 2014, when most U.S.-led forces are scheduled to leave Afghanistan.
Hold on, so the Taliban want the US forces, that they are supposedly fighting, to stay past 2014? Very interesting. This also goes along with older videos I've seen of Afghans claiming that Bush was arming the Taliban in the mountains.
2012.12.6 Hamid Karzai; US, NATO Behind Afghan 'Insecurity' (NBC, youtube.com):
Afghan President Hamid Karzai had some harsh words for the United States and NATO during a Thursday interview with NBC News.
Karzai accused the U.S. of violating its strategic partnership agreement with Afghanistan, and he indicated that "part of the insecurity [in Afghanistan] is definitely coming to us from terrorism and the attacks of the Taliban." He continued with a criticism of the U.S., adding, "part of the insecurity is coming to us from the structures that NATO and America created in Afghanistan."
The Afghan leader singled out "the private security firms" and "the contractors that they promoted at the cost of the Afghan people."
Karzai also partly blamed the insecurity in Afghanistan on "the way they [the U.S., NATO] behaved with the Afghan people and the anger that that has caused in the Afghan people and the resulting insecurity." He added, "there is a very strong perception that some of that insecurity is intentional."
The U.S. actions that stirred anger to which he was likely referring include an incident in which Marines were shown in a video urinating on the corpses of Taliban fighters. After the video came to light, Karzai called the actions of those involved "inhuman." Two Marines were criminally charged for their actions. In another incident, U.S. soldiers were implicated in the burning of copies of the Koran.
Karzai also leveled criticism at the U.S. in November, when he accused U.S. forces of violating a detainee transfer pact signed between the two countries.
New violence struck Afghanistan on Thursday when the country's intelligence chief, Asadullah Khalid, was wounded by a suicide bombing. The Taliban claimed responsibility for the attack.
NBC points out, per the U.N., that "in the first six months of 2012 alone, more than 3,000 civilians were killed or injured" in Afghanistan.
North Korea threatens U.S. with preemptive nuclear strike
By Jack Kim
SEOUL | Thu Mar 7, 2013 10:40am GMT
(Reuters) – North Korea threatened the United States on Thursday with a preemptive nuclear strike, raising the level of rhetoric while the U.N. Security Council considers new sanctions against the reclusive country.
North Korea has accused the United States of using military drills in South Korea as a launch pad for a nuclear war and has scrapped the armistice with Washington that ended hostilities in the 1950-53 Korean War.


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Invasive species, or plants and animals that have been introduced to an area, can cause harm when they establish themselves at the expense of native wildlife. These invaders pose a threat to native species by outcompeting them for resources like food and habitat that are necessary for survival. Often, these species expand their range and population numbers at such a rapid pace that landowners and wildlife managers struggle to contain their spread.
Brian Knox is President of Sustainable Resource Management, Inc., a natural resource consulting firm based out of Davidsonville, Md., that deals primarily with managing forest vegetation. Knox has seen success in combating invasive plants by implementing outside-the-box tactics. "A lot of people these days are getting more conscious about their herbicide usage," said Knox. "As a very small company, we're not afraid to try anything."
In 2007, Knox began unleashing a herd of goats—referred to as Eco-Goats—on areas overrun by invasive vegetation. The goats have proven to be a viable option for these problem areas, because they can go many places that people and machinery cannot reach, like steep slopes and hillsides. Additionally, because of the goats' mouth shape and digestive system structure, they are able to grind up seeds in a way that ensures seeds are not returned to the soil to resprout at the end of the digestive process.
A herd of about 30 goats can work through about a half-acre of dense vegetation in 3 to 4 days. "Goats are very good at biomass reduction," explained Knox. "Typically, a goat can eat about 25 percent of its bodyweight a day in green material. If you figure an average of 100 pounds, that's 25 pounds of vegetation going into every goat that's out there."
Although the goats are fond of invasive species like kudzu, porcelain berry, wine berry and mile-a-minute and are undeterred by thorns, they do not discriminate against native species. So before committing his goats to an area, Knox surveys each site to make sure the vegetation is appropriate. "A misapplied goat is every bit as bad as a chemical spill," said Knox. "You can do damage with a goat... I look for native species and ask, 'Is there more here to save than there is to get rid of?' If so, that's a terrible place for a goat."
Spending your day with a herd of goats may sound like fun, but managing the goats is hard work. "A lot of people think it's just sitting around and watching the goats, and boy, that would be a great job. And then you talk to them about how it took me two days to get the fence up and I'm soaked through to my socks by eight in the morning," said Knox. "But clearly there's something that I really love about this. And it's the educational aspect of it, seeing people's eyes light up while watching the goats."
To view more photos, visit the Chesapeake Bay Program's Flickr page.
Images and video by Keith Rutowski
Text by Jenna Valente
An economic analysis from the U.S. Fish and Wildlife Service (USFWS) shows that federal investments in on-the-ground restoration can stimulate local economies, creating jobs and supporting small businesses.
With a focus on two of its habitat restoration programs—the Partners for Fish and Wildlife Program and the Coastal Program—the USFWS determined that for every federal dollar spent, $7 to $9 of restoration work happens on the ground and almost $2 of economic activity is gained by the state in which the work takes place.
Both of these nation-wide programs use federal and private funding to implement on-the-ground habitat restoration projects on public and privately owned land. According to the USFWS, the programs' impacts cut across two dimensions: first, their understood expertise and stable funding pulls in additional funding from other partners; second, the programs' spending creates work, generates tax revenues and stimulates local economies through paid wages and subsequent spending.
Image courtesy Margrit/Flickr
In Maryland, for instance, the Coastal Program has directed $1.4 million toward the eradication of nutria from marshes and wetlands. Introduced to the region in the mid-1940s, the invasive nutria has destructive feeding habits, pulling up plant roots that would otherwise hold valuable marshland in place. The Maryland Nutria Project, which is administered by the USFWS and brings federal, state and private partners together to trap and manage nutria, has created more than 55 jobs and generated $2.5 million in spending on Maryland's Eastern Shore.
"The Partners for Fish and Wildlife and Coastal programs are important drivers for creating employment," said USFWS Director Dan Ashe in a media release. "The benefits reach far beyond the local communities where these projects take place to provide national economic stimulus. At the same time, this restoration work provides benefits to all Americans by creating healthy natural areas, including shorelines, streams, wetlands and forests on privately owned lands."
I enjoy eating fish. I also enjoy catching them. And after learning about the impact that a couple of recent invaders have had on the Chesapeake Bay, I've added two new fish species to my catch list.
About a year ago, I sat in a meeting (which I do a lot) and listened to a presentation on yet another threat to the Chesapeake Bay's aquatic environment. This threat came in the form of the blue and flathead catfish, the finfish equivalent to the invasive zebra mussel that has upset the aquatic ecosystem of the Great Lakes.
Blue catfish are considered "apex predators"; they sit at the top of the food chain. They consume not only other finfish, but shellfish as well. They have no predators. Introduced to Virginia's James, Rappahannock and York rivers as a sport fish in the 1960s, '70s and '80s, they have multiplied and extended their reach into other parts the Bay. Apparently, they are here to stay.
Recently, the Maryland Department of Natural Resources (DNR) launched a public awareness campaign to promote a "catch and cook" movement in place of the usual "catch and release" program. The agency is working with restaurants and fish processors to market these catfish. During the campaign's April kick-off event, we were treated to samples of what these demons of the deep might taste like. I went back for seconds (several times). This is a great source of protein for those who rely on fishing for both sustenance and subsistence. And while catfish are bottom feeders, I've been assured that, as long as we eat fish that are smaller than 32 inches, there shouldn't be any concern about the bioaccumulation of toxins. Good to know.
The Bay has become home to other invasive fish, as well. We all remember the northern snakehead! While this critter isn't as pervasive as the blue catfish, it is another species that has pushed portions of the Bay ecosystem out of balance. But let me recommend a snakehead ceviche. If you can't beat 'em, eat 'em! Bon apetit!
Note: The opinions expressed above are those of the author and do not necessarily reflect U.S. EPA policy endorsement or action.
The Maryland Department of Natural Resources (DNR) has launched a state-wide campaign to teach citizens about the impact of blue and flathead catfish and encourage anglers to remove the invasive species from local rivers and streams.
Native to the Mississippi, Missouri and Ohio river basins, blue catfish were introduced to the James, Rappahannock and York rivers in the 1970s and '80s as a sport fish. Flathead catfish were introduced to the James in the 1960s for the same reason. Over time, the natural movement and purposeful introduction of the fish into new waters have hastened their establishment in Chesapeake Bay tributaries.
This concerns scientists, who fear the fast-growing and long-lived blue catfish, in particular, could impact the region's ecologic and economic resources. Because of its opportunistic feeding habits, the blue catfish has become an apex predator, disrupting the structure of the Bay ecosystem and eating up critical aquatic species.
Indeed, "gut content analyses" of the fish have found American shad, Atlantic menhaden, freshwater mussels and blue crabs in their stomachs. Peyton Robertson, director of the National Oceanic and Atmospheric Administration's Chesapeake Bay Office and chair of the Chesapeake Bay Program's Sustainable Fisheries Goal Implementation Team, compared the blue catfish to a Bengal tiger, noting that the fish eats "just about anything."
"If left unchecked, [blue catfish] could, as top predators, start to impact other parts of our ecosystem," Robertson said.
But its eradication isn't feasible, and experts believe the invasive fish is here to stay. So managers hope to mitigate their spread and minimize their impact on native fish.
With support from the Bay Program, DNR has established more than 150 signs at water access points and kiosks around the state to help anglers identify, catch and keep the species, while Maryland Seafood has escalated its efforts to market the fish to restaurants and boost consumer demand.
"[Humans] are great at overfishing things," said Maryland Seafood Marketing Director Steve Vilnit. "And [the blue catfish] is a species that we want to overfish."
From the Field: Monitoring Blue Catfish in Virginia's James River from Chesapeake Bay Program on Vimeo.
In 2012, the Sustainable Fisheries Goal Implementation Team established an Invasive Catfish Task Force, which has drafted seven recommendations to help manage both blue and flathead catfish.
Every day, non-native plants, pests and diseases are introduced to the United States from around the world. These invaders have the power to out-compete native species, causing damaging effects to ecosystems and local economies, and the Chesapeake Bay is no stranger to these obtrusive critters.
The Bay is a vacation destination, shipping hub and home to more than 17 million people, a combination that puts it at high risk for invasive species introduction. Typically, non-native species travel to new areas by hitching rides on trade ships, travelers' luggage and recreational vehicles. Although not all invasive species are a threat, it is important to know which ones can and have caused widespread damage. Use the list below to identify 10 invasive species in the Bay watershed.
Image courtesy rbairdpccam/Flickr
10. Blue Catfish. The blue catfish is a large, smooth-skinned fish with a bluish gray body and whisker-like barbells around its mouth. It is native to the Mississippi, Missouri, and Ohio river basins but was introduced to the James, Rappahannock, and York rivers during the late 20th century. Blue catfish are considered invasive because of their active reproduction rates and large appetite for native fish and shellfish species. The fish feeds mainly on shad, menhaden, blue crab and river herring and has few natural predators that can prevent it from out-competing native species. Blue catfish can grow up to 100 pounds, live up to 20 years and are thought to make up 75 percent of the fish biomass in some portions of the Bay.
9. Mute Swans. The mute swan is a large, aggressive bird that is native to northern and central Eurasia. It was introduced to North America in the mid-1800's to add ambiance to parks and ponds, but individuals quickly escaped, set up nesting territories and the population spread. A single mute swan can consume up to 20 pounds of submerged aquatic vegetation daily, threatening important native aquatic plants. Mute swans are one of the most aggressive waterfowl species in the world, especially when nesting or brooding. They have been known to chase off, injure and even kill native waterfowl species.
Image courtesy of Chrisdetmer/Flickr
8. Zebra Mussels. The zebra mussel is a tiny bivalve with zebra-like stripes and a triangular shell. It lives in freshwater lakes, rivers and reservoirs in parts of the Bay watershed. It was introduced to the Great Lakes region in the 1980s, most likely via ballast water from a European ship, and quickly spread throughout the United States. Once introduced to a waterway, the zebra mussel attaches itself to hard surfaces and can produce millions of offspring annually. Zebra mussels compete with native bivalves, fish and invertebrates for plankton and are responsible for the drastic decline of native clam, mussel and oyster populations in some areas.
Image courtesy of Bernd Loos/Flickr
7. Nutria. The nutria is a large, brown, semi-aquatic rodent that looks like a beaver and lives in marshes and wetlands on the Delmarva Peninsula and other parts of the Bay watershed. Native to South America, the nutria was introduced in Maryland to Blackwater National Wildlife Refuge in 1943 for fur farming. Escaped animals soon began to reproduce, bearing up to three litters of four offspring each year, spreading rapidly. In Maryland, nutria is the greatest threat to salt marsh habitat because it eats sediment-holding plants and causes significant erosion.
6. Phragmites. Phragmites is a perennial plant with feathery plumes at the top of tall, stiff stalks. It grows in wetlands, along roadsides and along shorelines throughout the Bay watershed. Although its origin is unclear, it is widely distributed across Europe, Asia, Africa, North America and Australia. It was introduced to North America inadvertently in the 19th century from the ballasts of Eurasian trade ships. Phragmites crowd out native plants by creating tall, dense stands in wetland habitats.
Image courtesy of Lisa Roukis/Flickr
5. Purple Loosestrife. Purple loosestrife is a perennial plant with spikes of bright purple flowers that bloom in mid-to late summer. Native to Europe and Asia, the plant was both accidentally and intentionally introduced in the 18th and 19th centuries. Ship ballasts unintentionally carried the tiny seeds to North America while others planted it for its aesthetic value and healing properties. The plant is considered an invasive because it quickly establishes itself in wetlands, crowding out native plant species and producing up to 2 million seeds per year with no known natural predators.
Image courtesy of U.S. Department of Agriculture/Flickr
4. Emerald Ash Borer. The emerald ash borer is a green, shiny beetle that lives on ash trees in certain parts of Virginia, Maryland and Pennsylvania. Native to Asia, the emerald ash borer was discovered outside of Detroit in 2002 and made its way to the Bay watershed in 2003 when a Michigan nursery shipped ash trees to Maryland. Adult beetles cause little damage to ash trees but larvae feed on the inner bark, disrupting the dispersion of water and nutrients throughout the tree.
Image courtesy of Smithsonian Environmental Research Center/Flickr
3. Chinese Mitten Crab. The Chinese mitten crab is a light brown crustacean with a distinctive pair of hairy, white-tipped claws. Native to East Asia and a member of LaFondation's top 100 worst invasive alien species list, it has recently been found in small numbers in the Bay. In abundance it not only competes with native species for resources and threatens populations through predation but also damages fishing industries by feeding on fish in nets and damaging nets and other equipment. It is also known to erode soft sediment banks, dykes and costal protection systems through excessive burrowing.
2. Veined Rapa Whelk. The rapa whelk is a large, predatory marine snail that inhabits the lower Bay. It is native to the Sea of Japan and the Bohai, Yellow and East China seas in Asia. It was first discovered in the Bay in 1998 by a trawl survey group from the Virginia Institute of Marine Science (VIMS) and is believed to have been transported in larval form through ballast water from trade ships. It poses a threat to the Bay because it preys on native bivalves like clams, oysters and mussels, which are vital to the region's economy and ecosystem. It feeds by wrapping itself around the hinge of its prey's shell, then feeding between the openings.
Image courtesy of Sergey Yeliseev/Flickr
1. European Gypsy Moth. The European gypsy moth is one of the most destructive pests that has ever been introduced to North America. Moth larvae gorge themselves on the foliage of shrubs and trees, leaving the plants bare and susceptible to disease and damage from other pests. The gypsy moth was accidentally introduced to the United States in Medford, Mass., in 1869, by a professor conducting silk research. It has been established in parts of eastern North America for more than a century.