text:
Research Article | Open Access
Systemic Infection of Nicotiana benthamiana with Potato virus X Nanoparticles Presenting a Fluorescent iLOV Polypeptide Fused Directly to the Coat Protein
Plant virus-based nanoparticles can be produced in plants on a large scale and are easily modified to introduce new functions, making them suitable for applications such as vaccination and drug delivery, tissue engineering, and in vivo imaging. The latter is often achieved using green fluorescent protein and its derivatives, but the monovalent fluorescent protein iLOV is smaller and more robust. Here, we fused the iLOV polypeptide to the N-terminus of the Potato virus X (PVX) coat protein, directly or via the Foot-and-mouth disease virus 2A sequence, for expression in Nicotiana benthamiana. Direct fusion of the iLOV polypeptide did not prevent the assembly or systemic spread of the virus and we verified the presence of fusion proteins and iLOV hybrid virus particles in leaf extracts. Compared to wild-type PVX virions, the PVX particles displaying the iLOV peptide showed an atypical, intertwined morphology. Our results confirm that a direct fusion of the iLOV fluorescent protein to filamentous PVX nanoparticles offers a promising tool for imaging applications.
1. Introduction
The functionalization of plant virus-based nanoparticles is an emerging field, facilitated by the well-characterized genomic sequence and modular organization of many different plant viruses [1–3]. Plant viruses come in various shapes and sizes with different surface properties, allowing researchers to pick the most suitable virus for a given application. The structures of many plant viruses are known to atomic or near-atomic resolution [4–7]. The advantages of plant virus-based nanoparticles include their ability to self-assemble with precise symmetry and polyvalency, their monodispersity and stability under a range of conditions, their inherent safety (biocompatibility and noninfectivity in mammals), and the rapid, simple and scalable production that can be achieved by molecular farming in plants [8]. None of these properties can be matched by synthetic nanoparticles.
Potato virus X (PVX) is the type member of the genus Potexvirus (family Alphaflexiviridae) and is often used as a vector for heterologous gene expression in plants [9–18]. The complete 6.4 kb genomic RNA sequences of several PVX strains are known [19–21]. The plus-strand RNA genome has a 5′-methylguanosine cap and is polyadenylated at the 3′-terminus [22, 23]. Each PVX particle comprises ~1270 coat protein (CP) subunits helically arranged to form filamentous, flexible virions encapsidating the viral genomic RNA. The C-terminus of each CP subunit is located within the PVX particle whereas the N-terminus is exposed on the surface, providing an ideal site for the presentation of peptides and proteins [4, 24–28].
Genetic modification or chemical conjugation can be used to tailor the surface properties of plant viruses allowing them to be engineered, for example, as drug delivery vehicles, tissue scaffolds, and electronic devices [1, 8, 29–32]. Furthermore, fluorescence-labeled virus nanoparticles can be used for biomedical imaging [33–39]. Such labeling is usually achieved by the conjugation of chemical dyes to functional groups on the virus surface after particle assembly. Several studies have shown that PVX particles carrying molecular contrast agents such as Alexa Fluor647 can efficiently penetrate solid tumors and accumulate inside [40–42]. However, chemical conjugation results in a limited number of tagged CP subunits, whereas the genetic fusion of a fluorescent protein to the CP allows denser labeling [35]. The latter strategy can be challenging because the size and biochemical properties of the fusion protein may interfere with particle assembly, stability, or infectivity. Single amino acids or peptides can be fused directly to the N-terminus of the PVX CP, whereas longer peptides that prevent particle assembly require the presence of wild-type PVX CPs to avoid steric hindrance, resulting in the formation of hybrid particles containing both wild-type CP and the fusion protein. This is generally achieved by inserting the Foot-and-mouth disease virus (FMDV) 2A sequence between the transgene and the cp sequence, yielding a CP fusion protein, free target protein, and wild-type CP due to a ribosomal skip during translation (the overcoat method) [43–45]. The biochemical properties of peptides and proteins fused to the CP can also affect virus particle assembly, stability, or functionality (e.g., systemic spreading), including the number of serine/threonine [46] or tryptophan residues [47], and the isoelectric point (pI) [47, 48]. A PVX-mCherry overcoat system for in vivo tumor imaging in mouse models was recently reported [37].
Although fluorescent proteins such as mCherry and green fluorescent protein (GFP) are routinely used as reporters for molecular interactions, they are dependent on pH and oxygen, and their large size (at least ~240 amino acids) and complex structure make them more difficult to express [49–51]. The recently discovered monomeric fluorescent protein iLOV (light, oxygen, or voltage sensing) was engineered from the LOV2 domain of Arabidopsis thaliana phototropin 2 and can be fused to the movement protein of TMV for quantitative biological imaging applications [52–55]. The iLOV sequence is approximately half the size of mCherry thus reducing the genetic load on the recombinant viruses, and its reversible photobleaching properties make it ideal for the analysis of virus movement in plants [52, 55, 56]. Here, we compared the behavior of PVX-iLOV particles created by either direct fusion or the use of the FMDV 2A sequence, focusing on their long-distance movement and propensity for systemic infection and the morphology of the modified particles compared to wild-type PVX.
2. Materials and Methods
2.1. Construction of Plant Viral Vectors
The PVX-based vectors we used to produce recombinant particles displaying the iLOV polypeptide are shown in Figure 1(a). The iLOV sequence was amplified from source vector pSC1001a (Dr. S. Chapman, The James Hutton Institute, Dundee, Scotland) using the primers listed in Table 1 and was transferred to pCR2.1-TOPO as previously described [57]. An iLOV fragment was isolated from this intermediate vector using NheI and BspEI and was transferred to vector pTCXIIc [37] either as a direct fusion (replacing the mCherry and FMDV 2A genes) or as fusion via the FMDV 2A sequence (replacing only the mCherry gene). The iLOV sequence was also inserted into pPVX-N-mCherry-G4S-CP (our unpublished data) to create an iLOV-CP fusion via a (Gly4Ser)3 linker (G4S). The integrity of the resulting vectors pPVX-iLOV-CP, pPVX-iLOV-2A-CP, and pPVX-iLOV-G4S-CP was tested by PCR and sequencing. The vectors were propagated in Escherichia coli strain DH5α ready for the inoculation of Nicotiana benthamiana plants as previously described [57].
2.2. RNA and Protein Analyses
Total RNA was isolated from systemically infected N. benthamiana leaves using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and 3 μg RNA samples were treated with 3 U DNaseI (Thermo Fisher Scientific) as recommended. The corresponding cDNA was produced using M-MLV Reverse Transcriptase RNaseH Minus Point Mutant (Promega, Madison, USA). We mixed 1 μg of the DNaseI-treated RNA with 0.5 μM of the oligo-dT primer for hybrid PVX particles and 27 μl DEPC-treated water. The mixture was heated to 80°C and then incubated for 10 min on ice to ensure primer annealing before adding 10 μl 5x M-MLV reaction buffer, 8.5 μl DEPC-treated water, 1 mM dNTPs, and 1 μl M-MLV. The cDNA synthesis reaction was carried out for 30 min at 40°C, 20 min at 45°C, 20 min at 50°C, 20 min at 55°C, and 20 min at 70°C. After reverse transcription, residual RNA was degraded with 1 μl 10 mg/ml RNaseA for 1 h at 37°C. PCR was carried out with primers TGB-fw and CX4 and the products were resolved by 1.2% agarose gel electrophoresis to confirm the integrity of the iLOV-CP and iLOV-2A-CP cDNAs.
RNA levels in PVX-infected leaves were determined by quantitative real-time PCR (qPCR). We mixed 5 μl of recombinant PVX cDNA samples with 0.3 μM each of primers CX4i and CX8, 10 μl 2x iQ™ SYBR Green® supermix (BioRad, Munich, Germany), and 3.8 μl water. The qPCR program comprised three steps: 3 min at 95°C for polymerase activation and DNA denaturation and then 40 cycles of 15 s at 95°C, 30 s at 55°C, and 45 s at 72°C measured with SYBR Green. Finally, melting curves were constructed from 55°C to 95°C in 0.5°C/2 s steps using primers for the cp (CX4i and CX8) and tgb (CX3i and CX9) as previously described [14]. Triplicate measurements were taken with samples from at least three different plants.
Proteins were extracted from systemically infected N. benthamiana leaves and were analyzed by SDS-PAGE [58] and western blot, with specific antibodies against iLOV and the PVX CP as previously described [57].
2.3. Confocal Microscopy
Systemic infection with recombinant PVX particles was visualized by confocal laser scanning microscopy using a Leica TCS SP instrument (Leica Microsystems, Wetzlar, Germany). We removed cm leaf pieces showing fluorescence and mounted them with the abaxial surface facing upwards onto glass microscope slides. An HCX PL APO water-corrected objective was used with a drop of water placed directly on the coverslip to image viral cell-to-cell movement across the epidermal cell layer. The excitation and detection wavelengths for iLOV were 476 nm and 510–550 nm, respectively. LCS software was used for image analysis, following import into Photoshop CS5 (Adobe Systems, Mountain View, USA) for contrast adjustments and overlay.
2.4. Purification of iLOV-Displaying Particles
Purified iLOV-CP and iLOV-2A-CP virus particles were prepared as described in the protocol published by the International Potato Centre (Lima, Peru) with modifications as previously described [13]. Centrifugation with a sucrose cushion was omitted and the pellet was directly resuspended after clarification. Subsequent centrifugation was carried out at 7800 ×g for 10 min at 4°C instead of 2000 ×g for 5 min at 4°C, and the second ultracentrifugation to sediment the virions was at least 3-4 h instead of 1 h. The final clarification step was also omitted.
2.5. Transmission Electron Microscopy
Immunosorbent electron microscopy (ISEM) was carried out with virus-containing plant extracts from systemically infected leaves or purified recombinant viruses. Pioloform-coated nickel grids (Plano, Wetzlar, Germany) were incubated on a drop of monoclonal antibody MAC58 against PVX (kindly provided by Professor L. Torrance, The James Hutton Institute) for 20 min at room temperature to capture the recombinant PVX particles. Immunogold decoration of captured or adsorbed particles with primary anti-PVX antibody or the anti-iLOV antiserum and secondary goat anti-rabbit (GAR) 15 nm gold conjugate (British BioCell, Cardiff, UK) and imaging was carried out as previously described [57].
3. Results
3.1. Vector Construction and Infection of Plants
Three different PVX-based expression constructs were prepared, joining the iLOV sequence to the 5′-end of the PVX cp gene using different strategies: (a) a direct fusion, (b) via a (Gly4Ser)3 linker, and (c) via the FMDV 2A sequence (Figure 1(a)). Following the inoculation of N. benthamiana plants separately with each vector, plants infected with pPVX-iLOV-2A-CP showed initial fluorescent spots at the inoculation site 4 days after inoculation (dpi) and systemic infection was observed 6 dpi, as revealed by the general green fluorescence. These plants also displayed more severe symptoms than typically observed for systemic infection with wild-type PVX, including mosaic patterns and dwarfing, and necrotic lesions appearing from 10 dpi onwards (Figures 1(c) and 1(e)). In contrast, plants infected with pPVX-iLOV-CP showed initial fluorescent spots at the inoculation site 8 dpi or later. The long-distance movement of the virus was significantly delayed: systemic infection was not observed until 17–21 dpi (Figure 1(d)). Plants inoculated with the pPVX-iLOV-G4S-CP vector did not show any signs of systemic infection (Figure 1(f)).
Quantitative real-time PCR (qPCR) was carried out to determine the levels of cp RNA in leaves from systemically infected N. benthamiana plants (Figure 2). Total RNA was isolated from leaves showing iLOV fluorescence, cDNA synthesis was carried out and the integrity of the modified PVX sequence was verified by a control PCR with primers TGB-fw and CX4, amplifying a region between the triple gene block protein 3 (tgbp3) and cp genes (Figure 2(a)). We then determined the relative levels of the tgbp3 and cp RNAs with the housekeeping gene PP2A used as a reference. The amount of ilov and cp RNA was compared to the amount of tgb3 RNA, which can be found in the PVX genomic RNA (gRNA) and subgenomic RNAs (sgRNAs) 1 and 2 but not sgRNAs 3. This allowed a direct correlation between the iLOV-(2A)-CP RNA level and the total amount of PVX RNA. The quantity of tgbp3 and cp RNA was correlated to PP2A mRNA and the relative amount of cp to tgb3 was calculated and compared. We observed higher relative cp RNA levels for the iLOV-2A-CP fusion than for the iLOV-CP direct fusion (Figure 2(b)). This was consistent with the rapid infection and severe symptoms in plants infected with pPVX-iLOV-2A-CP compared to the slower systemic infection with pPVX-iLOV-CP.
3.2. Analysis of Recombinant PVX Particles Displaying iLOV
Extracts were prepared from leaves containing the pPVX-iLOV-CP and pPVX-iLOV-2A-CP fusion proteins for analysis by SDS-PAGE and western blot. The iLOV-CP and iLOV-2A-CP fusion proteins were detected with the anti-PVX antibody and the anti-iLOV antiserum with molecular weights of 38.0 and 39.9 kDa, respectively, as expected (Figures 3(b) and 3(c) highlighted with asterisks). A 25 kDa band representing the wild-type PVX CP was also detected in the samples containing iLOV-2A-CP because fusion to the FMDV 2A sequence is known to cause a ribosomal skip (Figure 3(b)). However, the CP was also detected by the anti-iLOV antiserum (Figure 3(c)). The 13.5 kDa free iLOV protein (highlighted with an arrow) was also visible in the western blot probed with anti-iLOV antiserum (Figure 3(c)) as well as in the polyacrylamide gel under UV light (Figure 3(d)). Densitometric analysis of iLOV bands in the anti-iLOV western blot showed a ratio of approximately 7 to 3 for free iLOV to the iLOV-2A-CP fusion protein.
The ability of the fusion proteins to assemble into PVX particles was confirmed by electron microscopy. PVX particles were immunocaptured from leaf extracts using monoclonal antibody MAC58, detected with a primary anti-PVX antibody and decorated with GAR 15 nm gold conjugates (Figures 3(e)–3(h)). Typically, wild-type PVX particles are flexible nm structures (Figure 3(g)), but iLOV-CP and iLOV-2A-CP particles showed a different morphology (Figures 3(e) and 3(f)). These particles were of the same size as wild-type particles but were intertwined, resulting in a morphology similar to that described for PVX particles with N-terminal coat protein truncations [59].
3.3. Localization of iLOV within the Plant Cell
To gain insight into the cell-to-cell movement of hybrid PVX particles, leaves systemically infected with pPVX-iLOV-CP and pPVX-iLOV-2A-CP were studied by confocal laser scanning microscopy (Figure 4). The iLOV fluorescence was concentrated in certain areas of the cell wall, which potentially indicates the cell-to-cell movement of PVX-iLOV particles through the plasmodesmata. The fluorescence profile (Figures 4(c) and 4(f)) indicated that the virus uses these intercellular links to cross the plant cell wall [60, 61]. We also observed large viral replication complexes (VRCs) within cells, typically one per infected cell [62, 63]. In leaves infected with pPVX-iLOV-2A-CP, we detected free iLOV protein in the cytoplasm and in the nucleus, reflecting the ribosomal skip at the FMDV 2A sequence (Figure 4(f)). The fluorescence of the iLOV-CP direct fusion led to a more precise localization of the hybrid virus within the plasmodesmata.
3.4. Purification and Immunogold Labeling of Virus Particles
PVX particles displaying iLOV were purified with yields of 136–206 mg per kg of infected leaf material by adapting the purification protocol for wild-type PVX particles developed by the International Potato Centre. The different pooled fractions were tested for the presence of iLOV-CP and iLOV-2A-CP by western blot (Figure 5). Both the polyclonal anti-PVX antibody and the anti-iLOV antiserum detected some degradation products but also intact proteins with the anticipated molecular weights of the iLOV-CP and iLOV-2A-CP fusion proteins (Figures 5(b) and 5(c)). The iLOV-CP fusion protein is only detected in very low amounts with an anti-PVX antibody, probably due to the low amounts of purified particles and protein degradation. The anti-iLOV antiserum also detected bands of the wild-type PVX CP, and thus it was necessary to find out whether the antiserum was able to also detect wild-type PVX particles by electron microscopy. Purified iLOV-CP, iLOV-2A-CP, and wild-type PVX virions were directly adsorbed onto the grids (Figure 5(d)) or captured using monoclonal antibody MAC58 (Figure 5(e)). The fusion proteins were detected by the primary anti-iLOV antiserum and labeled with the secondary GAR 15 nm gold conjugate. We found that only PVX particles displaying the iLOV protein on the surface were labeled, not the wild-type PVX particles. Thus, scattered single gold particles are most likely background and not bound to particles. These experiments also confirmed that the heterologous fusion protein is accessible even if the recombinant virions are intertwined.
4. Discussion
The biological compatibility and multivalency of genetically and chemically addressable sites on virus CPs have resulted in a broad range of applications for plant virus nanoparticles. In this study, PVX-derived vectors (Figure 1(a)) were generated to present the monomeric fluorescent protein iLOV on the surface of PVX particles by fusing it to the 5′-terminus of the PVX CP directly, via a FMDV 2A sequence (overcoat), or via a sequence coding for a flexible glycine-serine-rich linker. The direct fusion (pPVX-iLOV-CP) and overcoat (pPVX-iLOV-2A-CP) constructs were able to achieve systemic infection whereas the linker construct (pPVX-iLOV-G4S-CP) was not, most likely due to size limitation [15, 48, 64, 65], and the latter was therefore excluded from further experiments. To exclude the possibility that the flexibility of the G4S-linker enables iLOV to move between the CP subunits and prevents particle assembly, we replaced the G4S linker with the more rigid AK3A linker (three copies of the sequence AKKKA). The plants showed neither green fluorescence in inoculated leaves nor systemic infection at 28 dpi (data not shown).
Systemic infection with pPVX-iLOV-CP was delayed and the infection symptoms were mild, whereas rapid systemic infection was established following inoculation with pPVX-iLOV-2A-CP and the infection symptoms were severe, including necrosis (Figures 1(d) and 1(e)). This may reflect the ability of the iLOV protein to promote the synthesis of reactive oxygen species [66]. Following inoculation with pPVX-iLOV-2A-CP, the iLOV polypeptide was overexpressed as a fusion protein but also as a free protein due to the overcoat principle, potentially triggering the accumulation of greater quantities of reactive oxygen species in these plants compared to those infected with pPVX-iLOV-CP (Figure 1(e)).
When certain peptides or proteins are fused to plant virus CPs, the assembly of the virion is sterically or electrostatically inhibited and systemic movement prevented [15, 48, 64, 65]. However, fusing the 113-amino-acid iLOV polypeptide directly to the N-terminus of the PVX CP still allows the assembly of hybrid virions which can move from cell-to-cell and achieve systemic infections via translocation through the vascular system. The iLOV polypeptide is much larger than the largest peptides that have been displayed by direct CP fusion in other plant viruses, for example, 23 amino acids for Tobacco mosaic virus (TMV) [64], 30 amino acids for Plum pox virus [67], and 50 amino acids for Cowpea mosaic virus [65, 68]. With the help of a 15-residue linker, a 133-amino-acid fragment of staphylococcal protein A has been displayed on the surface of TMV [69], whereas the largest peptide expressed on the surface of PVX until the current report was 14 amino acids in length [12]. Fusion peptides larger than 40 amino acids usually require the presence of the wild-type PVX CP to assemble into intact hybrid particles, which is most easily achieved by incorporating the FMDV 2A sequence [43] as shown by the successful display of the 45 kDa VP6 rotavirus capsid protein (397 amino acids) on PVX [11]. The ratio of fusion protein to wild-type CP cannot be predicted accurately [70, 71], although using different variants of 2A sequences provides a degree of control [71]. As a tool for the bioimaging of viral cell-to-cell movement, fluorescent proteins should be densely arrayed on the virus surface to achieve a bright signal, as shown for the iLOV-CP direct fusion but not for the iLOV-2A-CP construct, which also generated diffuse fluorescence mainly caused by the free iLOV polypeptide (Figure 4).
We investigated the cp RNA level in relation to the tgbp3 RNA by qPCR because the PVX tgbp3 sequence is found on the gRNA and all sgRNAs except that carrying the cp gene, whereas the cp sequence is found on the gRNA and all sgRNAs. The comparison of cp and tgbp3 RNA levels therefore provides an estimation of cp RNA levels with a built-in internal control. The level of iLOV-CP RNA was lower than that of iLOV-2A-CP RNA (Figure 2), explaining why the iLOV-CP particles spread more slowly through the plant. Our data suggest there are lower levels of sgRNA3 and thus lower levels of the iLOV-CP fusion protein, which can assemble to intact particles for long-distance movement. A certain ratio between the number of assembled particles and the quantity of free PVX CP [46] is necessary for viral cell-to-cell movement and long-distance transport [47, 61, 72–74]. This is why fewer iLOV-CP particles assembled compared to iLOV-2A-CP particles, thus reducing the viral load. These results are consistent with the observed delayed systemic spread of iLOV-CP particles and the rapid infection and severe symptoms caused by iLOV-2A-CP. However, the differences of sgRNA levels are unexpected, given that the iLOV sequence is incorporated into the viral genome in the same way in both constructs. The only difference is the 2A sequence introduced between the iLOV and CP sequences. The introduction of the 2A sequence in this construct therefore seems to be more favorable even for sgRNA levels.
Some PVX coat proteins with N-terminal extension peptides cannot form particles due to steric inhibition [43], the number of serine/threonine [46] or tryptophan residues [47], or the pI of the fusion peptide [47, 48]. If fusion peptides have a pI value in the range 5.2–9.2, the resulting particles can move systemically [47], although basic amino acids reduce the fitness of the virus resulting in the selection of better performing mutants [48]. Only ~13% of the amino acids in the iLOV peptide are positively charged and the iLOV-CP fusion has a pI of 5.8. There was no reversion to the wild-type PVX cp sequence at 34 dpi as reported for iLOV fusions to the TMV CP [57]. However, we observed proteolytic degradation of the purified iLOV-CP particles in western blots probed with the polyclonal anti-PVX antibody and anti-iLOV antiserum after particle purification (Figures 5(b) and 5(c)). The particles might be degraded during the purification process due to a lower stability of the particles in general. However, the extracts contained larger quantities of the fusion products than the virus purification (compare Figures 3(b) and 3(c) and 5(b) and 5(c)). In addition to protein degradation, we might have lost particles with a high ratio of iLOV-CP fusion to wild-type CP during the gradient purification. Particles with a greater proportion of fusion proteins accumulate in the gradient with a lower sucrose concentration and might be lost due to low visibility in the SDS gels of the gradient fractions. This could probably be prevented by adapting the purification protocol accordingly.
The presence of iLOV-decorated PVX particles in systemically infected leaves was confirmed by western blots and transmission electron microscopy (Figures 3 and 5). Wild-type PVX particles are filamentous rods, 515 nm in length—they are flexible but exist as discrete units. In contrast, the iLOV-CP and iLOV-2A-CP particles were similar in structure but the filaments tended to intertwine. A similar morphology was reported when PVX particles were assembled from N-terminally truncated CPs lacking codons 7–31 of the cp gene [59]. These results show that the N-terminus is required to modulate intramolecular and/or intermolecular interactions. Approximately 37% of the residues in the iLOV protein are hydrophobic, so intermolecular hydrophobic interactions may be responsible for the intertwined morphology.
Confocal laser scanning microscopy revealed that the recombinant iLOV-CP and iLOV-2A-CP particles were potentially able to pass between epidermal cells, as indicated by the presence of a fluorescent signal in the plasmodesmata (Figure 4). Similarly localized CPs with 2A fusions of fluorescent proteins have been reported in PVX movement studies [60, 61, 75]. We also observed one large fluorescent VRC per infected cell, which is frequently observed in established infections [60, 62, 63]. These so-called "virus factories" coordinate the infection processes [62]. Additional diffuse fluorescence was observed in epidermal cells infected with the pPVX-iLOV-2A-CP construct, representing the presence of free iLOV polypeptide. Free iLOV was also detected in western blots, with a ratio of 7 to 3 relative to the fusion protein. This leads to a high background of free iLOV in the cells, preventing the detailed analysis of CP localization. These findings emphasize the advantage of direct fusions of fluorescent proteins to the target protein.
Fluorescent proteins can provide insight into many cellular processes and interactions but also suffer from drawbacks such as their complex structure, large size, pH, and oxygen dependence [50, 51]. The iLOV polypeptide has spectral characteristics similar to GFP [76], but it is monomeric, has a lower molecular weight, is more photostable, and undergoes reversible photobleaching, the latter providing advantages for imaging applications [52, 55]. The presentation of the iLOV fluorescent protein on the surface of filamentous PVX offers a promising new imaging tool, particularly for the investigation of PVX infections. More specifically, these hybrid virions may be able to provide insight into the subcellular location of CP synthesis and the distribution and packaging of gRNA. The iLOV polypeptide can also be used for antiviral drug screening [77].
5. Conclusion
We have demonstrated that the 113-amino-acid fluorescent protein iLOV can be directly fused to the N-terminus of the PVX coat protein without the help of a flexible linker and is displayed on the particle surface. Because the iLOV protein has several advantages over other fluorescent proteins, it will be useful not only for the analysis of VRCs and long-distance virus movement, which currently relies on the production of overcoat structures, but also as a new platform for the molecular imaging of tumors. We recently reported an mCherry PVX overcoat system for tumor imaging in mouse models [37]. Our PVX-iLOV particles generated a strong fluorescent signal due to the dense array of iLOV polypeptides, offering a highly sensitive new modality for imaging applications.
Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.
The authors acknowledge the excellent technical assistance of Karolin Richter. They thank Dr. Sean Chapman (The James Hutton Institute, Dundee, Scotland) for the kind gift of the iLOV sequence and Professor John Christie (Institute of Molecular Cell and Systems Biology, University of Glasgow, Scotland) for the anti-iLOV antiserum. The authors thank Dr. Jens Tilsner for helpful discussions and Dr. Richard M Twyman for critically reading the manuscript. This research was funded by the Excellence Initiative of the German Federal and State Governments (Gefördert durch Mittel der Exzellenzinitiative des Bundes und der Länder).
- C. Lico, E. Benvenuto, and S. Baschieri, "The two-faced potato virus X: from plant pathogen to smart nanoparticle," Frontiers in Plant Science, vol. 6, article 1009, 2015. View at: Publisher Site | Google Scholar
- J. L. M. C. Geelen, A. Van Kammen, and B. J. M. Verduin, "Structure of the capsid of cowpea mosaic virus. The chemical subunit: molecular weight and number of subunits per particle," Virology, vol. 49, no. 1, pp. 205–213, 1972. View at: Publisher Site | Google Scholar
- P. J. Butler, "The current picture of the structure and assembly of tobacco mosaic virus," Journal of General Virology, vol. 65, part 2, pp. 253–279, 1984. View at: Google Scholar
- A. Kendall, W. Bian, A. Maris et al., "A common structure for the potexviruses," Virology, vol. 436, no. 1, pp. 173–178, 2013. View at: Publisher Site | Google Scholar
- T. Lin, Z. Chen, R. Usha et al., "The refined crystal structure of cowpea mosaic virus at 2.8 Å resolution," Virology, vol. 265, no. 1, pp. 20–34, 1999. View at: Publisher Site | Google Scholar
- X. Agirrezabala, E. Méndez-López, G. Lasso, M. A. Sánchez-Pina, M. Aranda, and M. Valle, "The near-atomic cryoEM structure of a flexible filamentous plant virus shows homology of its coat protein with nucleoproteins of animal viruses," eLife, vol. 4, Article ID e11795, 2015. View at: Publisher Site | Google Scholar
- M. G. Rossmann, "Structure of viruses: a short history," Quarterly Reviews of Biophysics, vol. 46, no. 2, pp. 133–180, 2013. View at: Publisher Site | Google Scholar
- A. M. Wen and N. F. Steinmetz, "Design of virus-based nanomaterials for medicine, biotechnology, and energy," Chemical Society Reviews, vol. 45, no. 15, pp. 4074–4126, 2016. View at: Publisher Site | Google Scholar
- S. Chapman, T. Kavanagh, and D. Baulcombe, "Potato virus X as a vector for gene expression in plants," The Plant Journal, vol. 2, no. 4, pp. 549–557, 1992. View at: Publisher Site | Google Scholar
- D. C. Baulcombe, S. Chapman, and S. Santa Cruz, "Jellyfish green fluorescent protein as a reporter for virus infections," The Plant Journal, vol. 7, no. 6, pp. 1045–1053, 1995. View at: Publisher Site | Google Scholar
- G. J. O'Brien, C. J. Bryant, C. Voogd, H. B. Greenberg, R. C. Gardner, and A. R. Bellamy, "Rotavirus VP6 expressed by PVX vectors in Nicotiana benthamiana coats PVX rods and also assembles into viruslike particles," Virology, vol. 270, no. 2, pp. 444–453, 2000. View at: Publisher Site | Google Scholar
- K. Uhde, R. Fischer, and U. Commandeur, "Expression of multiple foreign epitopes presented as synthetic antigens on the surface of Potato virus X particles," Archives of Virology, vol. 150, no. 2, pp. 327–340, 2005. View at: Publisher Site | Google Scholar
- K. Uhde-Holzem, V. Schlösser, S. Viazov, R. Fischer, and U. Commandeur, "Immunogenic properties of chimeric potato virus X particles displaying the hepatitis C virus hypervariable region I peptide R9," Journal of Virological Methods, vol. 166, no. 1-2, pp. 12–20, 2010. View at: Publisher Site | Google Scholar
- C. Dickmeis, R. Fischer, and U. Commandeur, "Potato virus X-based expression vectors are stabilized for long-term production of proteins and larger inserts," Biotechnology Journal, vol. 9, no. 11, pp. 1369–1379, 2014. View at: Publisher Site | Google Scholar
- R. L. Toth, S. Chapman, F. Carr, and S. S. Cruz, "A novel strategy for the expression of foreign genes from plant virus vectors," FEBS Letters, vol. 489, no. 2-3, pp. 215–219, 2001. View at: Publisher Site | Google Scholar
- A. M. Zelada, G. Calamante, M. de la Paz Santangelo et al., "Expression of tuberculosis antigen ESAT-6 in Nicotiana tabacum using a potato virus X-based vector," Tuberculosis, vol. 86, no. 3-4, pp. 263–267, 2006. View at: Publisher Site | Google Scholar
- K. Uhde-Holzem, M. McBurney, B. D. B. Tiu et al., "Production of immunoabsorbent nanoparticles by displaying single-domain Protein A on Potato Virus X," Macromolecular Bioscience, vol. 16, no. 2, pp. 231–241, 2016. View at: Publisher Site | Google Scholar
- E. S. Mardanova, E. A. Blokhina, L. M. Tsybalova, H. Peyret, G. P. Lomonossoff, and N. V. Ravin, "Efficient transient expression of recombinant proteins in plants by the novel pEff vector based on the genome of potato virus X," Frontiers in Plant Science, vol. 8, article 247, 2017. View at: Publisher Site | Google Scholar
- M. J. Huisman, H. J. Linthorst, J. F. Bol, and J. C. Cornelissen, "The complete nucleotide sequence of potato virus X and its homologies at the amino acid level with various plus-stranded RNA viruses," Journal of General Virology, vol. 69, part 8, pp. 1789–1798, 1988. View at: Google Scholar
- B. E. Orman, R. M. Celnik, A. M. Mandel, H. N. Torres, and A. N. Mentaberry, "Complete cDNA sequence of a South American isolate of potato virus X," Virus Research, vol. 16, no. 3, pp. 293–305, 1990. View at: Publisher Site | Google Scholar
- K. G. Skryabin, A. S. Kraev, S. Y. Morozov et al., "The nucleotide sequence of potato virus x RNA," Nucleic Acids Research, vol. 16, no. 22, pp. 10929-10930, 1988. View at: Publisher Site | Google Scholar
- N. Sonenberg, A. J. Shatkin, R. P. Ricciardi, M. Rubin, and R. M. Goodman, "Analysis of terminal structures of RNA from potato virus X," Nucleic Acids Research, vol. 5, no. 7, pp. 2501–2512, 1978. View at: Publisher Site | Google Scholar
- S. Y. Morozov, L. I. Lukasheva, B. K. Chernov, K. G. Skryabin, and J. G. Atabekov, "Nucleotide sequence of the open reading frames adjacent to the coat protein cistron in potato virus X genome," FEBS Letters, vol. 213, no. 2, pp. 438–442, 1987. View at: Publisher Site | Google Scholar
- J. Sober, L. Jarvekulg, I. Toots, J. Radavsky, R. Villems, and M. Saarma, "Antigenic Characterization of Potato Virus X with Monoclonal Antibodies," Journal of General Virology, vol. 69, no. 8, pp. 1799–1807, 1988. View at: Publisher Site | Google Scholar
- L. A. Baratova, N. I. Grebenshchikov, E. N. Dobrov et al., "The organization of potato virus X coat proteins in virus particles studied by tritium planigraphy and model building," Virology, vol. 188, no. 1, pp. 175–180, 1992. View at: Publisher Site | Google Scholar
- L. A. Baratova, N. I. Grebenshchikov, A. V. Shishkov et al., "The topography of the surface of potato virus X: Tritium planigraphy and immunology analysis," Journal of General Virology, vol. 73, part 2, pp. 229–235, 1992. View at: Publisher Site | Google Scholar
- M. A. Nemykh, A. V. Efimov, V. K. Novikov et al., "One more probable structural transition in potato virus X virions and a revised model of the virus coat protein structure," Virology, vol. 373, no. 1, pp. 61–71, 2008. View at: Publisher Site | Google Scholar
- L. Parker, A. Kendall, and G. Stubbs, "Surface features of potato virus X from fiber diffraction," Virology, vol. 300, no. 2, pp. 291–295, 2002. View at: Publisher Site | Google Scholar
- G. P. Lomonossoff and D. J. Evans, "Applications of plant viruses in bionanotechnology," in Plant Viral Vectors, vol. 375 of Current Topics in Microbiology and Immunology, pp. 61–87, Springer, Berlin, Germany, 2011. View at: Publisher Site | Google Scholar
- J. N. Culver, A. D. Brown, F. Zang, M. Gnerlich, K. Gerasopoulos, and R. Ghodssi, "Plant virus directed fabrication of nanoscale materials and devices," Virology, vol. 479-480, pp. 200–212, 2015. View at: Publisher Site | Google Scholar
- J. F. Steele, H. Peyret, K. Saunders et al., "Synthetic plant virology for nanobiotechnology and nanomedicine," Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, vol. 9, no. 4, Article ID e1447, 2017. View at: Publisher Site | Google Scholar
- N. P. Montague, E. C. Thuenemann, P. Saxena, K. Saunders, P. Lenzi, and G. P. Lomonossoff, "Recent advances of cowpea mosaic virus-based particle technology," Human Vaccines & Immunotherapeutics, vol. 7, no. 3, pp. 383–390, 2011. View at: Publisher Site | Google Scholar
- F. M. Brunel, J. D. Lewis, G. Destito et al., "Hydrazone ligation strategy to assemble multifunctional viral nanoparticles for cell imaging and tumor targeting," Nano Letters, vol. 10, no. 3, pp. 1093–1097, 2010. View at: Publisher Site | Google Scholar
- K. L. Lee, L. C. Hubbard, S. Hern, I. Yildiz, M. Gratzl, and N. F. Steinmetz, "Shape matters: The diffusion rates of TMV rods and CPMV icosahedrons in a spheroid model of extracellular matrix are distinct," Biomaterials Science, vol. 1, no. 6, pp. 581–588, 2013. View at: Publisher Site | Google Scholar
- J. D. Lewis, G. Destito, A. Zijlstra et al., "Viral nanoparticles as tools for intravital vascular imaging," Nature Medicine, vol. 12, no. 3, pp. 354–360, 2006. View at: Publisher Site | Google Scholar
- M. Manchester and P. Singh, "Virus-based nanoparticles (VNPs): Platform technologies for diagnostic imaging," Advanced Drug Delivery Reviews, vol. 58, no. 14, pp. 1505–1522, 2006. View at: Publisher Site | Google Scholar
- S. Shukla, C. Dickmeis, A. S. Nagarajan, R. Fischer, U. Commandeur, and N. F. Steinmetz, "Molecular farming of fluorescent virus-based nanoparticles for optical imaging in plants, human cells and mouse models," Biomaterials Science, vol. 2, no. 5, pp. 784–797, 2014. View at: Publisher Site | Google Scholar
- A. M. Wen, S. Shukla, P. Saxena et al., "Interior engineering of a viral nanoparticle and its tumor homing properties," Biomacromolecules, vol. 13, no. 12, pp. 3990–4001, 2012. View at: Publisher Site | Google Scholar
- A. Niehl, F. Appaix, S. Boscá et al., "Fluorescent Tobacco mosaic virus-derived bio-nanoparticles for intravital two-photon imaging," Frontiers in Plant Science, vol. 6, article 1244, 2016. View at: Publisher Site | Google Scholar
- S. Shukla, A. L. Ablack, A. M. Wen, K. L. Lee, J. D. Lewis, and N. F. Steinmetz, "Increased tumor homing and tissue penetration of the filamentous plant viral nanoparticle potato virus X," Molecular Pharmaceutics, vol. 10, no. 1, pp. 33–42, 2013. View at: Publisher Site | Google Scholar
- S. Shukla, A. M. Wen, N. R. Ayat et al., "Biodistribution and clearance of a filamentous plant virus in healthy and tumor-bearing mice," Nanomedicine, vol. 9, no. 2, pp. 221–235, 2014. View at: Publisher Site | Google Scholar
- S. Shukla, R. D. Dorand, J. T. Myers et al., "Multiple administrations of viral nanoparticles alter in vivo behavior—insights from intravital microscopy," ACS Biomaterials Science and Engineering, vol. 2, no. 5, pp. 829–837, 2016. View at: Publisher Site | Google Scholar
- S. S. Cruz, S. Chapman, A. G. Roberts, I. M. Roberts, D. A. M. Prior, and K. J. Oparka, "Assembly and movement of a plant virus carrying a green fluorescent protein overcoat," Proceedings of the National Acadamy of Sciences of the United States of America, vol. 93, no. 13, pp. 6286–6290, 1996. View at: Publisher Site | Google Scholar
- M. L. L. Donnelly, L. E. Hughes, G. Luke et al., "The 'cleavage' activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring '2A-like' sequences," Journal of General Virology, vol. 82, part 5, pp. 1027–1041, 2001. View at: Publisher Site | Google Scholar
- M. L. L. Donnelly, G. Luke, A. Mehrotra et al., "Analysis of the aphthovirus 2A/2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'," Journal of General Virology, vol. 82, part 5, pp. 1013–1025, 2001. View at: Publisher Site | Google Scholar
- C. Betti, C. Lico, D. Maffi et al., "Potato virus X movement in Nicotiana benthamiana: new details revealed by chimeric coat protein variants," Molecular Plant Pathology, vol. 13, no. 2, pp. 198–203, 2012. View at: Publisher Site | Google Scholar
- C. Lico, F. Capuano, G. Renzone et al., "Peptide display on Potato virus X: molecular features of the coat protein-fused peptide affecting cell-to-cell and phloem movement of chimeric virus particles," Journal of General Virology, vol. 87, part 10, pp. 3103–3112, 2006. View at: Publisher Site | Google Scholar
- K. Uhde-Holzem, R. Fischer, and U. Commandeur, "Genetic stability of recombinant potato virus X virus vectors presenting foreign epitopes," Archives of Virology, vol. 152, no. 4, pp. 805–811, 2007. View at: Publisher Site | Google Scholar
- T. Drepper, T. Eggert, F. Circolone et al., "Reporter proteins for in vivo fluorescence without oxygen," Nature Biotechnology, vol. 25, no. 4, pp. 443–445, 2007. View at: Publisher Site | Google Scholar
- J. A. Gawthorne, L. E. Reddick, S. N. Akpunarlieva et al., "Express your LOV: An engineered flavoprotein as a reporter for protein expression and purification," PLoS ONE, vol. 7, no. 12, Article ID e52962, 2012. View at: Publisher Site | Google Scholar
- N. C. Shaner, G. H. Patterson, and M. W. Davidson, "Advances in fluorescent protein technology," Journal of Cell Science, vol. 120, part 24, pp. 4247–4260, 2007. View at: Publisher Site | Google Scholar
- J. M. Christie, K. Hitomi, A. S. Arvai et al., "Structural tuning of the fluorescent protein iLOV for improved photostability," The Journal of Biological Chemistry, vol. 287, no. 26, pp. 22295–22304, 2012. View at: Publisher Site | Google Scholar
- A. Mukherjee, J. Walker, K. B. Weyant, and C. M. Schroeder, "Characterization of flavin-based fluorescent proteins: an emerging class of fluorescent reporters," PLoS ONE, vol. 8, no. 5, Article ID e64753, 2013. View at: Publisher Site | Google Scholar
- M. G. Khrenova, A. V. Nemukhin, and T. Domratcheva, "Theoretical characterization of the flavin-based fluorescent protein iLOV and its Q489K mutant," The Journal of Physical Chemistry B, vol. 119, no. 16, pp. 5176–5183, 2015. View at: Publisher Site | Google Scholar
- S. Chapman, C. Faulkner, E. Kaiserli et al., "The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection," Proceedings of the National Acadamy of Sciences of the United States of America, vol. 105, no. 50, pp. 20038–20043, 2008. View at: Publisher Site | Google Scholar
- A. Cheuk and M. Houde, "A rapid and efficient method for uniform gene expression using the barley stripe mosaic virus," Plant Methods, vol. 13, article 24, 2017. View at: Publisher Site | Google Scholar
- J. Röder, R. Fischer, and U. Commandeur, "Adoption of the 2A ribosomal skip principle to tobacco mosaic virus for peptide display," Frontiers in Plant Science, vol. 8, article 1125, 2017. View at: Publisher Site | Google Scholar
- U. K. Laemmli, "Cleavage of structural proteins during the assembly of the head of bacteriophage T4," Nature, vol. 227, no. 5259, pp. 680–685, 1970. View at: Publisher Site | Google Scholar
- S. Chapman, G. Hills, J. Watts, and D. Baulcombe, "Mutational analysis of the coat protein gene of potato virus X: effects on virion morphology and viral pathogenicity," Virology, vol. 191, no. 1, pp. 223–230, 1992. View at: Publisher Site | Google Scholar
- J. Tilsner, O. Linnik, M. Louveaux, I. M. Roberts, S. N. Chapman, and K. J. Oparka, "Replication and trafficking of a plant virus are coupled at the entrances of plasmodesmata," The Journal of Cell Biology, vol. 201, no. 7, pp. 981–995, 2013. View at: Publisher Site | Google Scholar
- S. Santa Cruz, A. G. Roberts, D. A. M. Prior, S. Chapman, and K. J. Oparka, "Cell-to-cell and phloem-mediated transport of potato virus X: The role of virions," The Plant Cell, vol. 10, no. 4, pp. 495–510, 1998. View at: Publisher Site | Google Scholar
- O. Linnik, J. Liesche, J. Tilsner, and K. J. Oparka, "Unraveling the structure of viral replication complexes at super-resolution," Frontiers in Plant Science, vol. 4, article 6, 2013. View at: Publisher Site | Google Scholar
- J. Tilsner, O. Linnik, K. M. Wright et al., "The TGB1 movement protein of potato virus X reorganizes actin and endomembranes into the X-body, a viral replication factory," Plant Physiology, vol. 158, no. 3, pp. 1359–1370, 2012. View at: Publisher Site | Google Scholar
- M. Bendahmane, M. Koo, E. Karrer, and R. N. Beachy, "Display of epitopes on the surface of tobacco mosaic virus: Impact of charge and isoelectric point of the epitope on virus-host interactions," Journal of Molecular Biology, vol. 290, no. 1, pp. 9–20, 1999. View at: Publisher Site | Google Scholar
- C. Porta, V. E. Spall, K. C. Findlay, R. C. Gergerich, C. E. Farrance, and G. P. Lomonossoff, "Cowpea mosaic virus-based chimaeras: Effects of inserted peptides on the phenotype, host range, and transmissibility of the modified viruses," Virology, vol. 310, no. 1, pp. 50–63, 2003. View at: Publisher Site | Google Scholar
- X. Shu, V. Lev-Ram, T. J. Deerinck et al., "A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms," PLoS Biology, vol. 9, no. 4, Article ID e1001041, 2011. View at: Publisher Site | Google Scholar
- M. R. Fernández-Fernández, J. L. Martínez-Torrecuadrada, J. I. Casal, and J. A. García, "Development of an antigen presentation sytem based on plum pox potyvirus," FEBS Letters, vol. 427, no. 2, pp. 229–235, 1998. View at: Publisher Site | Google Scholar
- C. Porta, V. E. Spall, J. Loveland, J. E. Johnson, P. J. Barker, and G. P. Lomonossoff, "Development of Cowpea Mosaic Virus as a High-Yielding System for the Presentation of Foreign Peptides," Virology, vol. 202, no. 2, pp. 949–955, 1994. View at: Publisher Site | Google Scholar
- S. Werner, S. Marillonnet, G. Hause, V. Klimyuk, and Y. Gleba, "Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A," Proceedings of the National Acadamy of Sciences of the United States of America, vol. 103, no. 47, pp. 17678–17683, 2006. View at: Publisher Site | Google Scholar
- G. A. Luke, H. Escuin, P. D. Felipe, and M. D. Ryan, "2A to the fore–research, technology and applications," Biotechnology & Genetic Engineering Reviews, vol. 26, no. 1, pp. 223–260, 2009. View at: Publisher Site | Google Scholar
- E. Minskaia, J. Nicholson, and M. D. Ryan, "Optimisation of the foot-and-mouth disease virus 2A co-expression system for biomedical applications," BMC Biotechnology, vol. 13, article 67, 2013. View at: Publisher Site | Google Scholar
- R. L. S. Forster, D. L. Beck, P. J. Guilford, D. M. Voot, C. J. Van Dolleweerd, and M. T. Andersen, "Thecoat protein of white clover mosaic potexvirus has a role in facilitating cell-to-cell transport in plants," Virology, vol. 191, no. 1, pp. 480–484, 1992. View at: Publisher Site | Google Scholar
- O. N. Fedorkin, A. G. Solovyev, N. E. Yelina et al., "Cell-to-cell movement of potato virus X involves distinct functions of the coat protein," Journal of General Virology, vol. 82, part 2, pp. 449–458, 2001. View at: Publisher Site | Google Scholar
- O. N. Fedorkin, A. Merits, J. Lucchesi et al., "Complementation of the movement-deficient mutations in potato virus X: Potyvirus coat protein mediates cell-to-cell trafficking of C-terminal truncation but not deletion mutant of potexvirus coat protein," Virology, vol. 270, no. 1, pp. 31–42, 2000. View at: Publisher Site | Google Scholar
- K. J. Oparka, A. G. Roberts, I. M. Roberts, D. A. M. Prior, and S. S. Cruz, "Viral coat protein is targeted to, but does not gate, plasmodesmata during cell-to-cell movement of potato virus X," The Plant Journal, vol. 10, no. 5, pp. 805–813, 1996. View at: Publisher Site | Google Scholar
- J. M. Christie, "Phototropin blue-light receptors," Annual Review of Plant Biology, vol. 58, pp. 21–45, 2007. View at: Publisher Site | Google Scholar
- X. Dang, S. Chalkias, and I. J. Koralnik, "JC virus-iLOV fluorescent strains enable the detection of early and late viral protein expression," Journal of Virological Methods, vol. 223, pp. 25–29, 2015. View at: Publisher Site | Google Scholar
Copyright © 2018 Juliane Röder et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


text:
You are on eastlansinginfo.org, ELi's old domain, which is now an archive of news (as of early April, 2020). If you are looking for the latest news, go to eastlansinginfo.news and update your bookmarks accordingly!
news from 2013 - 2020
You are on eastlansinginfo.org, ELi's old domain, which is now an archive of news (as of early April, 2020). If you are looking for the latest news, go to eastlansinginfo.news and update your bookmarks accordingly!
As ELPD and MSU feared might happen, unruly crowds gathered in East Lansing after Michigan State's 61-51 loss to Texas Tech in the Final Four on Saturday night. The official police report has now come out, showing 23 arrests in total, including 22 misdemeanors and one felony. Two street signs and one police van were vandalized.
According to a news release from the City, "the ELFD and ELPD and assisting agencies responded to 10 small fires in the City. These fires consisted of small pieces of furniture and there were no significantly sized crowds gathered around these fires."
There were 22 citations in total for open alcohol and disorderly conduct, with the majority of those coming from the large gathering near Cedar Village. The felony arrest was for damage to a police transport van, by a prisoner according to Deputy Police Chief Steve Gonzalez.
In preparation for trouble, ELPD and MSUPD brought in reinforcements from the Michigan State Police, Ingham County Sheriff's Office, Eaton County Sheriff's Office, Meridian Township Police Department, Lansing Police Department, and the Clinton County Sheriff's Office.
The cavalry also showed up, in the form of the volunteer mounted unit.
The Cedar Village area once again represented the epicenter. By the time the buzzer sounded at the end of the second half of the game, there was already a large police presence there. Media outlets from all over the state came to witness events, and throughout the night, a news helicopter could be heard hovering in the sky.
Shortly after the game ended, MSU students began congregating at the corner of River Street and Waters Edge Drive to voice their frustration with the results. At one point, the crowd began chanting, "we want couches, we want couches."
They did not get their wish. But the largest crowd – estimated by police to number between 1,200 and 1,500 people – hooted and hollered.
The group then began moving north to Grand River where they shut down the entire road for about a half hour.
Drivers, for the most part, didn't seem to care. Some even got out of their cars and filmed, and some joined in with the students. There was no expectation that the road would be opening anytime soon.
Some in the crowd found and ripped down a "road closed" sign and began waving it at the cars that were stopped.
One person decided to tear down the street sign at the corner of Grand River Avenue and Woodmere Street. Once it was down, others passed it around like a championship trophy.
According to the City's news release, "Soon after assembling on Grand River Avenue, the crowd began to tear down street signs and throw glass bottles into the air, effectively making the gathering a civil disturbance." Police began to encircle the group. After about a half hour, they were dispersed without the use of chemical sprays.
In Lubbock, Texas – the home of Texas Tech – things got uglier. Cars were overturned, couches were set ablaze and the crowd had to be dispersed with tear gas.
What happened there was more like what happened in East Lansing in 1999. That year, as the Lansing State Journal recently recounted, following MSU's loss to Duke in the Final Four an estimated five to ten thousand people took to the streets to voice their frustration. One hundred and thirty-two people were arrested, including 71 students, and between $250,000 and $500,000 worth of property was damaged.
Afterwards, the Ingham County prosecutor's office subpoenaed reporters' recordings in an attempt to identify suspects. The LSJ and other news organizations refused to comply and the Michigan Supreme Court ultimate sided with them.
In 1999, East Lansing's City Council passed a law aimed at punishing those who might take photos of fires set at these kinds of "civil disturbances." In 2016, East Lansing Judge Andrea Larkin declared the law unconstitutional.
Update: this article was updated to reflect changes in the nature of arrests.
eastlansinginfo.org © 2013-2020 East Lansing Info

subset: commoncrawl


text:
Before the likes of Robin Roberts, Anderson Cooper and others, Thomas Roberts made history as the first network anchor to come out.
That was back in the day when he worked at CNN in Atlanta. He moved away not long afterwards but now he and his partner of 19 years, Patrick Abner, are back in the area. Last July he returned to the city to be an anchor on the Atlanta affiliate CBS46, where he can be seen evenings covering news and issues that excite him, like politics.
Ironically, being on air was never the plan for Roberts. While he was at Western Maryland College, majoring in communication and minoring in journalism, he was required to take an internship. He missed the deadline for The Baltimore Sun but was allowed to intern at a TV station. The station was CV3 — Prestige Cablevision — and Roberts was hooked from day one. "I fell in love with the immediacy of it all," he admits. "I was supposed to be there two hours the first day and I was there eight. They could hardly get me to leave. From that day forward, I changed the trajectory of what I wanted to do. I knew I wanted to be a reporter, but I wanted to try TV." He landed a job at the station after school.
He moved around a lot including San Diego, California; Lincoln, Nebraska; Fort Myers, Florida; and Portsmouth, Virgina. By this time he had an agent and was looking to make a move to Philadelphia. A job was available there — which he interviewed for — but when he returned home, CNN made contact with his agent. Roberts knew he had to pursue it.
On the way to the interview, he stepped in a wad of bright green gum outside and was a bit freaked out. The incident turned out to be a tension breaker, though, and after a Friday interview he received an offer the following Monday.
That was in 2001 and Roberts stayed at CNN for six years. "It was a great time. It was right after the AOL/Time Warner merger and they were looking to do new things. 9/11 had just happened and everybody was turned upside in terms of what news meant to people," he said.
In 2006, while at the National Lesbian and Gay Journalists Association convention in Miami, Roberts made the decision to come out as gay publicly. Getting to that point was scary, however. "I was petrified of doing it. There were no examples of successful out broadcast journalists, and no real path. Until Don Lemon came out, it was a lonely island," he said.
Roberts left Atlanta the following year, and after stints in Los Angeles with Entertainment Tonight and The Insider, he wound up at MSNBC in 2010, where his show Live with Thomas Roberts won an Emmy Award for its coverage of the Supreme Court decision on marriage equality. Now he's back in Atlanta doing what he loves and in a familiar territory. CBS46 is known for being gay-friendly and last year the station broadcast the Atlanta Pride parade in its entirety, with plans to do so again this year. "It's nice to know that LGBTQ stories here are looked at with the same equity (as others)."
When he's not at the station, Robert enjoys working out, traveling and spending time renovating his new Morningside house. He already feels at home. "New York was exciting but we needed a change," he says. "Because of our comfort level here, it made it easier to come back to Atlanta."


text:
Ciao tutti,
SO today was the third evening of the Institute's centenary cultural programme with the skilled businessman and speaker Lorenzo Bini Smaghi on the outlook for the European economy and financial markets. Smaghi gave a great overall perspective of the economic climate of today. From Donald Trump's potential policies to the consequences of Italy's tradition of family businesses, he illustrated the major economic questions facing Italy, Europe and our world as a whole.
Speaking to my classmates after the talk, Tim commented, "it was a very stimulating discussion about the current challenges for the European and global economy with a strong focus on Italy in particular." Whilst Michael expressed that "it was a fascinating talk that provoked more questions than answers" and "it deserved more time to tie up loose ends!"
Meanwhile, my first WISDOM tooth has surfaced and I don't think it's a coincidence that this has happened whilst being exposed to such passionate and interesting speakers!
In other news, Jules and I indulged in a manicure today at the lovely Wave beauty salon on Via Santo Spirito.
I particularly loved the lighting hence the photos!! It was also SO great to be able to use my Italian in a very casual, friendly situation with the wonderful Italian woman who made our nails beautiful once more. It's so nice to not feel like a total tourist and engage with local people.
Today I won't be posting an idiom but instead the funny linguistic difference between how Italians and English speakers avoid using profanities…
Now whilst we english say Sugar! the Italians instead say Cavolo! literally meaning cabbage!
I hope you've enjoyed today's post as much as I'm enjoying my new Italian lifestyle!!
Mills X


text:
Amazon Prep is officially AMZ PREP INC., a Mississauga, Ontario, based warehouse and shipping office dedicated to helping Amazon Sellers make it big!
We love what we do and like to be associated with successful sellers and story makers.
As an Amazon Seller, count us in your journey for making it big!! We love what we do and like to be associated with successful sellers and story makers. We are a group of motivated individuals with combined experience of advanced supply chain management.
We started this journey after a complete understanding of the entire value chain of Amazon FBA. We come with an expertise of providing you innovative solutions and allow you to continue your journey of product search without having an iota of doubt about the logistics involved. We are passionate about our work and aim to put our efforts in the right direction in alignment with the needs of customers like you.
Amazon Prep Location:
1200 Aimco Blvd., Unit 6, Mississauga, Ontario
What does AMZ Prep Do?
We excel in prepping and shipping your products to Amazon FBA in Canada or any other carrier which transports your products to Amazon US FBA, feel free to add us in your journey as an effective and reliable partner.
Call us. We'll respond to your needs promptly and personally. Try us now!!
Who are the Founders of AMZ Prep?
Adi leads our Sales & Marketing Operations. Adi helps customers understand our offerings and perpetually helps us understand our individual customer needs. In our business, we have three types of people. Those who make things happen those who watch it happen & those who wonder what happened. Which one are you?
Girish Modha, B.Eng. (Electronic Systems Engineering)
Girish leads our Prep Operations. He's a fan of simplification and you can sense it as you are having a brief interaction with him. He believes in good, better, best. Never let it rest. Till your good is better and your better is best. Hope you agree with him, as we all do.
David is a T-shaped Professional. He has deep knowledge in trademarks with broad education and experience in AI, business law, business management, eCommerce, engineering, entrepreneurship, HR, litigation, marketing, and patents.


text:
Parthiv Patel's dream season capped off by Mumbai Indians' IPL 2017 triumph
Mumbai Indians won their record third Indian Premier League (IPL) 2017 title on Sunday and Parthiv Patel believes it was the unity and humility that helped them win.ipl-2017 Updated: May 22, 2017 23:35 IST
Mumbai Indians' unsung hero Parthiv Patel described the Indian Premier League (IPL) 2017 as dream season after finishing as top-scorer for the champions.
Parthiv Patel is one of the few players that have been part of the Indian Premier League setup since its inaugural season. He produced a career-best performance after aggregating 395 runs.
Read | ICC Champions Trophy: Rohit Sharma has no issues opening for India
"I am happy I have finished as the team's highest run-getter but the most important thing is to win trophies and being a part of this set-up," Parthiv said on Monday, a day after helping Mumbai Indians win a dramatic final against Rising Pune Supergiant at Hyderabad.
"Every time we've had a good opening partnership we've (MI) always gone on to get the runs. A good start especially in the 20-over format helps the bigger players like Rohit and Pollard to play freely," he said.
Mumbai won their record third IPL title and Parthiv believes it was the unity and humility that helped them win.
Read | Jos Buttler takes off towel while celebrating Mumbai Indians' IPL 2017 title win
"Winning the IPL to top it off was very special. It's a long tournament and you have to make sure the team stays together for that period. Rohit and the coaching staff did a fantastic job on that front through the season. They made sure our feet were on the ground," said the wicketkeeper.
Parthiv also picked his Test comeback and the Ranji Trophy triumph with Gujarat as the other defining moments this season. He led Gujarat to victory over Mumbai in the Ranji final in January with a match-winning 143 in the second innings.
Read | IPL 2017 top 10 moments: Two hattricks, lowest total of 49 and a triple crown
"Winning the Ranji Trophy is every player's dream. It was sweeter winning it for Gujarat," said Parthiv.
Parthiv's call up in the Test series against England late last year marked his comeback after eight long years. "I'm really happy to have made the comeback. It has always been a dream for me to play in a Test before I finish off," said the 32-year-old.
Despite consistent performances, Parthiv missed out on selection for the Champions Trophy, but he is not losing sleep over it. "Honestly, I don't worry about selection anymore. I am old enough and been around for years. I don't play cricket to get selected."


text:
Good works done
Special investigation team of District Police Chief arrested the accused person who involved in several crime cases .Read More
The Janamaithri police have shown a fine example of humanitarian gesture by giving away a newly constructed house for aged and ailing Bhargavi Amma and her mentally unstable daughter-27-08-2019.Read More
Two persons were arrested in connection with the smuggling of ganga by the shadow police - 07-08-2019Read More
Kidnapping and possible blackmailing was sucessfully foiled by timely interference of Adoor PoliceRead More
The finantial assistance by Janamaithri Police of Moozhiyar Police Station,the aged Srilankan couple could return to there home land even after the expiry of their Visa Validity.Read More
The accused person of around 10 cheating cases was arrested by Pathanamthitta Police. He was absconding during the bail period.Read More
Shadow police of District Police Chief Pathanamthitta seized banned tobacco products from diffrent places intented to sell to the students.Read More
Keezvaipur Police Station Crime No. 328/2015, 400/2015, 399/2016
Keezvaipur Police arrested KaliMuthu an accused involved in several cases of Keezvaipur Police Station.Read More


text:
4k H.265 Pro NVR5000
16/32 Channels Network Video Recorder
The 4K H.265 NVR 5000 is a device which supports 16/32 channels network video recorder (NVR). It can perfectly manage ONVIF and third-party cameras. It is designed with high compatibility and stability features. The basic difference between Mini and pro 4K H.265 NVR is of the number of channels. This device can handle up to 32 channels and 4K Mini NVR can handle only 8. The device is compatible and highly stable with the other systems. High-efficiency video coding is used in NVR 5000 series. The bandwidth requirement for the device is about 160 Mbps with 12V DC supply.
- Flexible in almost all weather condition and works under -10 degrees to +55 degrees of Celsius.
- Powerful Decoding and Recording performance. It supports decoding 4-CH 4K UHD, 16-CH 1080P and simultaneous recording up to 32-CH 5MP high-resolution cameras.
- It comes with various interfaces to be configured with.
- The most trusted one is Cohesive Technologies. They are the most ideal one.
Apart from these, you can find manifold other smarter features and we at Cohesive Technologies give you the best options.
Features & Benefits
- 4K Video Viewing Experience: Milesight 4K H.265 Pro NVR 5000 Series introduces a whole new more smooth operation interface.
- Powerful Decoding and Recording Performance: This series can support synchronously decoding 4-CH 4K UHD, 8-CH 4MP and 16-CH 1080P network cameras, and synchronously recording up to 32-CH 5MP high resolution cameras.
- Versatile Interfaces: Milesight 4K H.265 Pro NVR 5000 Series comes with a whole array of ports such as an HDMI port, a VGA port, Alarm I/O and a LAN port, etc.
Solutions & Success Stories
Explore Cohesive Technologies Solutions that are tailor-designed for each and every industrial verticals.
Hotels & Hospitality
Auto & Transportation
Media & Retail
MILESIGHT 4K H.265 Pro NVR 5000 PRODUCT INTRODUCTION
Get to know the features, capabilities and unique benefits of Milesight 4K H.265 Pro NVR 5000 Network Video Recorder.Watch the Video
Complementary Products


text:
20-something student and avid reader, reading and reviewing anything and everything under the sun (although YA definitely dominates)
Unravel Me (Shatter Me #2) by Tahereh Mafi
Release Date: 5th February 2013
Publisher: HarperCollins
Format: eBook
Pages: 348
Rating: 5.0/5.0
Summary from Goodreads:
it's almost
time for war.
Juliette has escaped to Omega Point. It is a place for people like her—people with gifts—and it is also the headquarters of the rebel resistance.
She's finally free from The Reestablishment, free from their plan to use her as a weapon, and free to love Adam. But Juliette will never be free from her lethal touch.
Or from Warner, who wants Juliette more than she ever thought possible.In this exhilarating sequel to Shatter Me, Juliette has to make life-changing decisions between what she wants and what she thinks is right. Decisions that might involve choosing between her heart—and Adam's life"
Usually I give myself longer to process a book before writing a review. But, 2 days after finishing this one, I need to get this review up to stay up to date with the schedule I wrote myself for the Christmas break, so here goes nothing. Unravel Me was amazing. Simply amazing.
It's been well over a year since I read Shatter Me, but Unravel Me transported me back to that world instantly. With the unique writing style that Mafi has employed in these books, how could it not? Merely pages into the book I'm asking myself Why did I wait so long to read this? Seriously, I don't understand why this wasn't at the top of my TBR, because Ignite Me definitely is. I just love how Mafi has employed this pseudo stream of conciousness for Juliet. You really get to understand Juliet better as a character because of it. You hear everything she's thinking, feel everything she's feeling through the poetic nature of the prose, understand the thoughts she's ashamed of having as they've been crossed out. It's nothing short of genius on Mafi's part. Everything is intensified, which makes this one of those books you struggle to put down.
Let's talk a bit about Warner, because he's one of my favourite characters by far. We learn a lot more about Warner in this novel. You get to see a lot more of the good, rather than the bad. He's very much a tortured soul - it's obvious that years of mistreatments have shaped him into who he is; there's a back story there just waiting to be told. I believe a lot more in Juliet and Warner's relationship than I do in Juliet and Adam's. Juliet and Adam always felt a bit instalovey for me; it seemed that the main reason Juliet was ever interested in him was because he was somehow immune to her touch. I think her relationship with Warner, on the other hand, shows a much more natural progression - he has to work a bit harder to win her over, so I'm much more inclined to want Warner with Juliet. Plus, that bedroom scene - lordy lordy bee.
Overall, Unravel Me was a very solid follow up to Shatter Me. This has a strong, action filled plot, a love triangle that I didn't hate, and a lot of new and interesting personalities were introduced. Plus Kenji; lets not forget about the awesomeness that is the witty, inappropriately acting yet serious when needed Kenji. And after that intense ending, I cannot wait to get my hands on Ignite Me early next year.


text:
we weren't able to find any production warehouse hiring now no background check jobs in Wallingford, Connecticut. let's try a different search to find you that perfect job.
Drop in and say "hi" at one of our branch locations.
for job seekers
for business
career resources
learning center
about randstad


text:
This is the first newsletter from the SUCCESS centre. Our intention is to inform about the activities in our project in a brief and easy-to-read way.
The newsletter will be published on an irregular basis, but at least four times a year.
SUCCESS News Letter No 1 May 2012
To be on our distribution list, please send an e-mail to firstname.lastname@example.com.


text:
Is there life beyond our planet? Astronomers have asked that question ever since we realized that there actually was something beyond our planet. Given the vastness of the universe, however, we're not likely to journey out and meet it for ourselves anytime soon. Instead, astronomers are searching for a way to bridge the vast distances of interstellar space and search for subtle signs of life on other planets from right here on Earth.
SETI has garnered attention for its far-reaching aim: t


text:
This week Desirai is joined her brother Lazarus and Samantha Olvera aka Lady Pwncess. They talk about Animal Crossing: Happy Home Designer, the World of Warcraft movie trailer, Disney discontinuing slave Leia merch, nutscapes and more.
Interested in supporting The Mommy Gamers? Earn exclusive rewards by becoming a patron on Patreon. Remember, you can subscribe to The Mommy Gamers podcast for FREE on iTunes and Stitcher or you can access The Mommy Gamers app through Podcast Box on iTunes or in the Amazon app store for Android. Don't forget to follow them on Twitter, and Facebook too!
Podcast: Play in new window | Download


text:
Rebuilding the local food system
VENUE CHANGE Due to demand, we've moved to: Biz Dojo, 115 Tory St, Wellington.
Our mission is to make local food convenient, affordable and fair everywhere.
We exist to help rebuild local food economies because we believe that changing the way we grow, process and distribute food is fundamental to solving the world's most pressing social and ecological problems.
Come and hear Pete Russell and James Samuel from Ooooby (Out Of Our Own Back Yards), talk about the crowd funding equity raise, how you can get your slice of the Ooooby pie and where we might be popping up next.
We've been working for the last 7 years to bring Ooooby from an exciting idea into a thriving organisation. Come along and hear the story so far and the vision moving forward.
Ooooby is ready to grow and scale - naturally. I'm delighted to have been part of the early conversations and see great things for this inspiring social enterprise.
115 Tory St, Te Aro, Wellington 6011, New Zealand
map Open in Google maps | Apple maps | Citymapper
An event page by James Samuel
Made with love in London
We ask for your email address so that we and the attendees have a way of contacting you.


text:
February 15–March 10, 2019
Friday and Saturday 7:30 p.m.
Sunday 2 p.m.
St. Bonaventure, 1625 W. Diversey Blvd., Chicago
(enter on Marshfield)
Editor's Note: Leigh Johnson shares the third of his works receiving its world premiere at Saint Sebastian Players (SSP) when Little Stones opens February 15. SSP premiered his play Lefties in 2013 and Lotto Fever in the Sucker State in 2014. Other Chicago productions of his work include Khe Sanh Bagman in 2000, Brother's Keeper in 2016 and A Prayer for the Sandinistas in 2017. He is an associate member of SSP and a founding member of Subtext Theater Company. In addition to being a Chicago-based playwright, he is a novelist and screenwriter. Picture This Post talked with Leigh for some insight into the play and the playwright.
(Picture This Post) What first attracted you to writing plays?
(Leigh Johnson) From a very early age, I knew I wanted to be a writer. As a child I was a voracious reader, and I found that after reading a story of some kind I couldn't wait to tell my family and friends about it. So it was this duality—not just the reading, but the need to relate it back (and usually in my own words)—that probably led to my becoming a playwright. It actually took me quite a while to figure this last part out, that the best avenue for telling my stories was for the stage, and not the short story form, which is what I mostly grew up reading. It really was the need to hearout loudthe story I was telling that made me a playwright.
Is this script one that sates that initial desire?
Absolutely. Of all the plays I've written, this one is probably the most lyrical. In the past I prided myself that my characters and plots involved real-life people with real-life problems. My characters did not wax poetic about the more esoteric aspects of life. But Little Stones came from a very different place, a place where the characters are struggling to find a deeper meaning for their existence, as opposed to fighting daily battles. And when your characters are so engaged, their hopes, fears, and yearnings come directly from the heart. And the language of the heart is poetry, which of course must be heard to be fully appreciated.
What inspired you to write this play?
Two years ago, my other theatre company, Subtext, was rehearsing another of my plays in an upstairs classroom at St. Bonaventure. (St. Bonaventure hasn't had a school at their facility since the 1980s, and the classrooms sit empty.) As it was, a couple of other small theatre companies were also renting rehearsal space, and I knew several of these other actors and people from past productions, as did a number of other actors. This is not unusual—the Chicago theatre community is very close knit. The combination of the old Catholic school classrooms—where children for decades began their formative years—starting at one end of the hallway and working their way down to the other—along with the concept of actors who in the past had been in plays with other actors present, were currently in another play, and in the future would probably be paired up yet again—really resonated with me. I—like a lot of people these days—am very interested in Buddhist thought and principles. And to me actors encountering other actors from past plays in the course of rehearsing a current play seemed almost an exact metaphor for twin souls and reincarnation: the idea that we have all lived many lives. And we will live other lives in the future, very probably with some of the same souls with whom we have previously been involved. The more I considered this, the more I realized there was a play to be had there.
Was the inspiration the historical elements or St. Bonaventure or both?
It really was both. Once I had the framework for the play, I was able to fold in the history of the parish and the neighborhood, as well as the significance of the 100-year anniversary of the great flu pandemic. Those two aspects really came together nicely and fit the overarching story quite well.
Who do you think would especially be drawn to this story?
First of all, anyone involved in Chicago theatre, especially the bare-bones skin-of-your-teeth storefront variety. This play is really a love letter to these people—the actors, directors, tech people, costumers, stage managers, etc. etc. etc. who toil away for no pay to follow their passion. It's no cliché to state that the phenomenon that is Chicago theatre has its beating heart in the storefronts and the church basements.
But in addition to theatre people, this play should especially be of interest to those for whom Buddhist principles and philosophy resonate deeply within their lives. For those who have found or are seeking relief from the noise and confusion of our 24/7 existence, those for whom meaning and mindfulness are more important than financial success and social status.
Is that why you were drawn to the story?
Yes, for both reasons. It would not surprise me at all to discover that some of my theatre compatriots and I have been through all of this before, in another time and another place. That is how close I feel to some of them.
What should people know about the history the play references, especially the 1918-19 flu pandemic?
That this was probably the first time a specific human disease was truly a worldwide phenomenon. Because of the massive troop movements from World War One, the virus spread from continent to continent, which really hadn't ever occurred before. So, for the first time, the world suddenly got smaller and what happened in rural Kansas had implications in Europe and beyond. That's kind of a big deal—that the 1918–19 flu pandemic heralded a change in human interconnectivity.
What is it like to watch a rehearsal and see your words come to life?
It's a pretty good high, but it's also humbling. Because when you see what the actors are doing with your words, how they are making them their own, taking ownership of them, you realize you only did the preliminary work. It is the director and actors who ultimately breathe life into the play, and to watch that happen slowly over the course of a number of rehearsals is quite remarkable. I suppose it's kind of like watching your kids grow up. At some point you ask in wonder, "Well, where did that come from?"
This is the third premiere of your work the Saint Sebastian Players have produced, along with two of your earlier works. What do you value about this partnership?
Most importantly, trust. When I bring a new work to SSP, I know the people are concerned only with making the play as good as it can be. That's it. There are no alternative political agendas, egotism, or destructive rivalries within the company. The sole focus, whether a new work or Shakespeare, is to put together a production team and a cast that will best serve the work. And then everyone pulls together and makes it happen. It really is quite remarkable in that they've been doing this now for 38 years—which in theatre years, well, is like 200 years, the way companies come and go.
Do you have ideas for your next play or plays?
Not specifically, no. But then, the well was dry when I was rehearsing that play in Classroom 101 at St. Bonaventure also. And within an hour of that epiphany…I had a new play. So you just have to trust that the Muse will not forsake you.
All photos by Eryn Walanka


text:
represents a mail folder associated with an active mail server connection.
- A MailFolder[…] object is always initially obtained from a MailServerConnection[…].
- Messages can be retrieved by position in a folder according to:
MailFolder[…][n] the n message from the beginning MailFolder[…][-n] the n message from the end MailFolder[…][m;;n] messages m through n MailFolder[…][{p1,p2,…}] a list of messages or spans MailFolder[…][All] all messages in the folder
- MailFolder[…][spec,MaxItems->max] specifies that only up to max items will be returned.
- MailFolder[…]["prop"] gives the specified property of a mail item.
- Possible properties include:
"Name" name of the folder "Path" complete path for the folder "MessageCount" count of messages not marked for deletion "FullMessageCount" count of messages, including ones marked for deletion "UnreadMessageCount" count of unread messages not marked for deletion "FullUnreadMessageCount" count of unread messages, including ones marked for deletion
Introduced in 2018


text:
Clearance - Final sale.
Wrap yourself up in our versatile new warm up top. Made of the same buttery soft high tech fabric and colors as the Grace, Odette and Margot Leotards. Mix and match ... you're going to want this top in onyx and quartz. longer sleeves with thumb holes flattering wrap around detailing elite performance fabric moisture wicking anti-bacterial technology Made in the USA in Orange County, CA


text:
Sunday, 8 September 2013
Colette Laurel Top
11 comments:
Love the fabric and the lace on the sleves. I once heard that you should smile right at the last min before the picture is taken as it looks more natural - not sure if it actually works though!ReplyDeleteReplies
Thanks for the tip - I'll give that a try next time!Delete
I love the trim on the sleeves too. Visiting from Brit Sewing Thursday linky.ReplyDeleteReplies
Thanks for popping by - glad you like the trim too!Delete
I've just started sewing skirts for my girls & can't wait to be able to make things for me too. Looks great :-)ReplyDeleteReplies
Thank you! You definitely need to make some treats for yourself as well as your girls - I've been sewing for a while but only just started dressmaking and it's great fun!Delete
Thanks for linking up this week on the Brit Sewing Thursday linky.ReplyDelete
I love the top you made and the trim on the sleeves is an absolutely gorgeous detail.Replies
- Anonymous24 September 2013 at 05:14
I think I have serious envy now! This top is fantastic! I love the lace bias binding (I've seen it on two different blogs today after having never seen it before...!).ReplyDelete
Taking photos really really sucks! I think you take really nice ones though and you are far braver than me (I skulk indoors rather than venturing outside!). I find that putting on happy music, trying not to stand up straight (I look so awkward when I do this!), and looking just to the side of the camera tends to help. I may not be the best person to give advice though because my photos are pretty bad...! ;-)Replies
Thank you, very kind of you to say so! It strange how it often seems to happen that you see something new for the first time and then suddenly it's everywhere isn't it?!Delete
Good tips on the photos, thanks - it's also good to know that I'm not the only one who feels awkward doing it, that alone makes it better if that makes any sense!


text:
John Brown's eldest, also John Brown, led his Pottawatomie Rifle company to Dutch Henry's tavern on April 21, 1856. They left their rifles behind, but close enough that if something happened they could arm themselves in a hurry. John Junior went into the tavern, which caused a stir as everyone recognized him and knew him as an antislavery militant, and asked if Judge Sterling Cato's court would enforce the laws of the United States or the laws of the bogus legislature. Cato saw that Brown and company hadn't come in packing heat, so he told them that the laws of the territorial legislature applied.
Junior took his answer and went outside, to where most of his company waited on the news. The men can't have taken it well. He came back inside the tavern toward the end of Cato's proceedings and spoke up, as he recounts to the Herald of Freedom
May it please the Court, I have a question in writing to propose to the court, an answer to which, would enlighten the citizens, and no doubt would be acceptable to the Grand Jury.
Cato did not take the interruption well. He told Brown to wait until he was finished and ignored Brown's note until then. Picking it up, he read
To the Court. Does this Court intend to enforce the enactments of the Territorial Legislature, so-called?
In Brown's words, Cato
took up the paper containing the question, and after looking at it, laid it down near the clerk, in a rather contemptuous manner, without making any reply whatever. The clerk then did the same thing, and also the marshal.
Stephen Oates, citing manuscript sources I don't have access to, expands on the contemptuousness. The judge read the paper and then threw it across the table. Brown adds that the clerk and marshal likewise read and discarded it. Cato would not have his court disturbed by theatrics, apparently.
Brown writes that he waited "awhile longer." Maybe he thought that if he did, Cato would oblige him after all. The court had finished its work, so answering a technical question of obvious relevance wouldn't be a great burden. Cato persisted in declining to play his part in Brown's drama.
Junior could take the hint and had a flair for the dramatic that day which no proslavery man would get in the way of. He stepped just outside the tavern and then declared, loudly so everyone inside could hear,
The Pottawatomie Rifle company will meet at the parade ground.


text:
Long Side Button Waistcoat Jacket
by Danielli
Sold Out
An easy to wear long waistcoat in camel, with a side button design. Throw this on for the layered look - easy to wear and super stylish - I particularly love the dropped shoulder giving a slight sleeve.
One Size 10/18
50% viscose 28% polyester 22% nylon
30 degree gentle wash
The UK Standard Delivery charge is £4.95 to anywhere in mainland UK. All orders will require a signature on receipt.
All items will be sent (under normal circumstances) within 2- 3 days of receipt of payment.
If you would like your order sent with Next Working Day delivery guaranteed (delivery before 5pm) the delivery charge is £8.50. This service is not available for guaranteed delivery on a Saturday.
For next working day deliveries orders need to be received by 2pm. Our standard delivery charge for UK Highlands, Islands, Isle of Man and Channel Islands is £9.95.
In the event that goods are not in stock at Danielli but are in stock at the manufacturer, delivery will be made as soon as the product is available. We will contact you to keep you up-to date.
You must notify us promptly and in any event within 48 hours of receipt of any goods if they appear to be damaged in transit. In those circumstances, you must also keep all packaging as this may be required when making a claim. Photographic evidence may be required.


text:
Stevey Newnez
Mon-Fri: 6AM
IG @steveynewnez | TWITTER @steveynewnez | FACEBOOK Stevey Newnez
Stevey Newnez is #TheFreakenDominirican from Your Hot Morning Crew, known for his 6:45a #SpanishWordoftheDay and Spanish Noticias in his #CalienteEnLaManana! He was actually raised in New Britain and is the official host of the Greater Hartford Latino Fest. He is also known for making appearances on some of your favorite TV Shows! On here, you'll get to see alot of his Fashion Highlights... Stevey believes in Fashion meeting Radio! Watch him as he breaks that "face for radio" stereotype, because "radio is more than just a voice" Stevey says!


text:
Dillion Harper loves to fuck and especially loves fucking LIVE! She can not wait to get Dannys hard cock deep in her wet pussy! Danny teases her letting her only have a taste but she craves more and soon they are fucking away hard. Dillion feels that large cock penetrate her deep as she cries out and cums! Danny is not done though fucking her more and harder until he is ready to give her bush a nice wet load!
2 year 9 month ago


text:
Emotions Conditionals Lesson
B2 level
Main Aims
To provide clarification and practice of the zero and first conditional in the context of emotions.
Subsidiary Aims
To provide fluency and accuracy speaking practice in mingling and group activities in the context of emotions.
Procedure (31-45 minutes)
-Ask the ss how their weekends were. Recap to last lesson by asking questions about what they remember. What did you talk about last lesson? (Emotions); What kind? (anger, dear, distress, etc.); How do people deal with these emotions? (Therapy); What were some of the types of therapy? (Destruction, music). -Have ss who were at the last lesson explain the topic to the ss who were not there in open pairs.
-Have ss do the first exercise on the handout (5A from book) and peer check. -Project the two categories with Powerpoint on the board. Have ss check that their answers were correct. -Using one sentence from each category (GS/FS), elicit the meaning of the sentences from the ss. Concept check. -Elicit the form from the ss: What is the same in these two sentences? (What/If); etc. Write the form as elicited underneath the sentences and leave on board. -Drill both forms with ss using one sentence from (GS) and one from (FS). Use back-chaining and focus on the contractions "I'll" and "we'll," and the weak form of "'ll."
-Have ss read the sentences and then do the listening exercise on the handout (6a from book). Have students peer check. Play a second time if necessary. -Monitor and provide direct feedback.
-Explain the third exercise on the handout (7a from book) to ss. Be clear that they must first match the sentences before they fill in the blanks. ICQ to be sure they understood/were listening. -Have ss do exercise and peer check. -Monitor during exercise and during peer check. Go over any numbers that were difficult together as a class. -Project answer key onto board and have ss check their answers with the answer key and each other.
-Hand out second handout and have ss do the exercise (1a from book). -Have ss peer check when finished. -Monitor and pick up points. -Go over any difficult numbers as a class (especially the last three, which are more difficult). -Give the ss the answer key and have them check their answers.
-Have ss make a circle with their desks. -Explain the game/activity and do an example with one or two students. Make it clear that when they have a "0," they must use the "1" conditional, and when they have a "1," they must use the first conditional. -Hand out the cut ups, and have the ss start the activity. -Monitor while they create their story.
-Provide feedback to the final activity, focusing on any problems with pronunciation and grammar. -Concept check the zero and first conditionals once more to be sure the ss now have a good grasp of them both and when they are used. -End class.


text: Worley and Bedard: NL Starting Pitchers by Dan Wade May 2, 2012 Most pitchers have four or five starts under their belt now that we're a month into the season, and while that's enough to start seeing some trends, it's also few enough that there is still some…fuzziness. I don't expect Madison Bumgarner and Roy Halladay to finish the season with strikeout rates below 7, for example, and on the other end of the spectrum, I'm not quite buying R.A. Dickey has generating the strikeouts at the same rate as Clayton Kershaw over the course of a full season. That all said, not all is smoke and mirrors, and finding those players now instead of a month from now could be the difference between surviving a midseason swoon and languishing at the bottom of the league. Here are two starters who have at the very least caught my eye as potential overachievers. Vance Worley Last season was a nice coming out party for Worley, who got his shot largely because of the injury to Joe Blanton, but there was some expectation of regression heading into the 2012 season. Instead, through five starts he has actually gotten a little better. His k-rate is the 15th best in the National League, and while K-rate can ebb and flow, Worley's improved rate doesn't look out of control. He struck out 8.13 per nine in the majors last year, and his improvement this year is tied to the fact that he's throwing his slider and curveball more often. Those pitchers are Worley's best both for generating whiffs and for producing groundballs. His ERA is artificially low at 1.97, a product of a strand rate that's closer to perfect than to league average, but neither his FIP nor his xFIP make it seem as though he's going to collapse into a below-average starter any time soon. On the positive side, I don't think Worley is going to finish the season as the Phillie who allows the most home runs; I think his 18.2 percent HR/FB rate will come down as well. As both go down, his ERA will come up, but fewer home runs will hopefully offset at least some of the runs that start to creep in. I don't see Worley as being a Brandon Morrow-esque pitcher where getting a good number of strikeouts comes at the cost of less-than-ideal rate stats. As long as he can keep the ball in the park, Worley looks like the type of pitcher who could be a strong SP3 candidate all season long. Erik Bedard Watching Bedard's first five starts, one gets the sense that in order to win games with any semblance of consistency Bedard is going to have to set the major league record for perfect games in a season. He allowed just 8 runs total in his first four starts and lost them all, finally recording his first win over the Braves last week. He'll never be the pitcher the Mariners thought they were trading for when they gave up Adam Jones, but that doesn't mean he doesn't have value, especially now that he has moved to the NL. I think his K-rate, which at 8.07 is actually slightly below his career average, is sustainable as he's facing opposing pitchers instead of DHs, but as he's also fighting the effects of age. His flyball rate is a little on the higher end, but with Jose Tabata and Andrew McCutchen patrolling the Pirates outfield, it's a vice that can be indulged for the time being. The biggest issue I have with him at the moment is that he's being a little inefficient and not working deep into games; he has made it out of the fifth inning just twice this season. It's not the worst trait a pitcher can have, but it does mean that if he gives up three runs or more, it'll hurt his ERA more than it would for someone who can reliably go at least six or seven innings. Bedard isn't a WHIP specialist, the walks have been part and parcel of his game for most of his career, but the strikeouts and solid ERA should make him a strong option at the back end of fantasy rotations. We hoped you liked reading Worley and Bedard: NL Starting Pitchers by Dan Wade! Please support FanGraphs by becoming a member. We publish thousands of articles a year, host multiple podcasts, and have an ever growing database of baseball stats. FanGraphs does not have a paywall. With your membership, we can continue to offer the content you've come to rely on and add to our unique baseball coverage. Support FanGraphs


text:
Rutgers Cancer Institute of New Jersey
195 Little Albany Street
New Brunswick, NJ 08903-2681
A clavicle fracture is a break in the collarbone. It connects the breastbone to the shoulder.
Copyright © Nucleus Medical Media, Inc.
This injury is caused by trauma from:
Babies can also get this injury from passing through the birth canal.
Things that can raise your risk are:
Large babies are at greater risk during birth.
Symptoms may be:
The doctor will ask about your symptoms and health history. The doctor will also ask how the injury happened. An exam will be done that will focus on the shoulder.
Images may be taken of your shoulder. This can be done with:
It may take three to ten weeks to heal. Newborns and most children do not usually need to have the pieces of the bone put back in position unless the broken ends are very far apart. Options may be:
The goals of treatment are to manage pain and support the bone as it heals. This may include:
Children's bones have growth plates that let bones grow and harden with age. A child with a fracture may need to be checked over time to make sure the bone heals the right way and keeps growing.
Some people may need surgery. Pins, a plate, or screws may be used to hold the bones in place as they heal.
Most fractures are due to accidents. They cannot be prevented.
American Orthopaedic Society for Sports Medicine
Ortho Info—American Academy of Orthopaedic Surgeons
Canadian Orthopaedic Association
Canadian Orthopaedic Foundation
Clavicle fracture—emergency management. EBSCO DynaMed website. Available at:https://www.dynamed.com/management/clavicle-fracture-emergency-management. Accessed September 24, 2019.
Murray IR, Foster CJ, et al. Risk factors for nonunion after nonoperative treatment of displaced midshaft fractures of the clavicle. J Bone Joint Surg Am. 2013 Jul 3;95(13):1153-1158.
Shoulder trauma (fractures and dislocations). Ortho Info—American Academy of Orthopaedic Surgeons website. Available at:https://orthoinfo.aaos.org/en/diseases--conditions/shoulder-trauma-fractures-and-dislocations/. Updated September 2007. Accessed September 24, 2019.
Last reviewed September 2019 by EBSCO Medical Review Board Warren A. Bodine, DO, CAQSM Last Updated: 9/24/2019


text:
Saturday, December 5, 2009
New Bundle of Joy
There will not be any new watch updates for awhile as I will likely be very busy with my new bundle of joy - a baby princess, who arrived on 03-Dec-2009. Would like to share this happy moment with my blog supporters. Thank you and see you folks around soon. Merry Christmas and a Happy New Year!
Posted by kohym
3 comments:
- Limau tambun said...
COngratulations on the new gift of love.
- December 5, 2009 at 11:51 PM
- Speedmaster said...
Wonderful, congrats!!!
- December 10, 2009 at 3:02 AM
- Unknown said...
Congratulations! :-) Looking forward to reading more watch articles frm u.
- December 23, 2009 at 7:05 AM
Subscribe to: Post Comments (Atom)