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Interferon-mediated host responses are of great importance for controlling influenza A virus infections. It is well established that the interferon-induced Mx proteins possess powerful antiviral activities toward most influenza viruses. Here we analyzed a range of influenza A virus strains for their sensitivities to murine Mx1 and human MxA proteins and found remarkable differences. Virus strains of avian origin were highly sensitive to Mx1, whereas strains of human origin showed much weaker responses. Artificial reassortments of the viral components in a minireplicon system identified the viral nucleoprotein as the main target structure of Mx1. Interestingly, the recently reconstructed 1918 H1N1 "Spanish flu" virus was much less sensitive than the highly pathogenic avian H5N1 strain A/Vietnam/1203/04 when tested in a minireplicon system. Importantly, the human 1918 virus-based minireplicon system was almost insensitive to inhibition by human MxA, whereas the avian influenza A virus H5N1-derived system was well controlled by MxA. These findings suggest that Mx proteins provide a formidable hurdle that hinders influenza A viruses of avian origin from crossing the species barrier to humans. They further imply that the observed insensitivity of the 1918 virus-based replicon to the antiviral activity of human MxA is a hitherto unrecognized characteristic of the "Spanish flu" virus that may contribute to the high virulence of this unusual pandemic strain.
Influenza A viruses pose a major health problem worldwide: thousands of people suffer from seasonal influenza A virus epidemics each year. Moreover, the virus has the potential to cause devastating pandemics (29). It is therefore important to understand the factors that determine influenza A virus virulence. Among them, polymerase activity, receptor specificity, and the cleavability of the hemagglutinin as well as the NS1 interferon (IFN) antagonist have previously been identified as important (27). In particular, the ability to down-regulate IFN production and/or action determines the pathogenic potential of some highly virulent strains to a large extent (7, 19, 35, 42).
The IFN system is a major component in innate immunity against viruses. IFNs help to limit virus propagation by inducing an antiviral state in potential target cells and by enhancing adaptive immune responses (40). They are known to induce hundreds of cellular genes, among them the Mx gene, which codes for the Mx protein with antiviral activity against influenza A viruses (10). Mx proteins belong to the dynamin superfamily of large GTPases and are found in many species, including fish, birds, and mammals (11).
The mechanisms by which Mx proteins exert their antiviral action are still not fully understood. The mouse Mx1 protein accumulates in the cell nucleus and inhibits primary transcription of influenza A virus, which occurs in the same subcellular compartment (22, 31), indicating that the viral ribonucleoprotein complex is a likely target structure of Mx1. Interference with this early step in the viral replication cycle should result in a dramatic inhibition of virus growth. Indeed, strong inhibition of influenza A virus replication by Mx1 has been observed in tissue culture experiments (37) and in experimentally infected Mx1+/+ mice that carry the Mx1 resistance gene (8, 9, 34, 44). IFN-regulated Mx genes are also present in humans. The human MxA protein inhibits the replication of influenza A virus and related orthomyxoviruses in cells and transgenic animals (5, 13, 31). In contrast to mouse Mx1, which resides in the cell nucleus, the cytoplasmic human MxA protein does not inhibit primary transcription of influenza A viruses but inhibits a subsequent step involved in genome amplification and secondary transcription (31). Whether primary or secondary transcription is affected depends on the subcellular localization. When MxA is moved into the nucleus by virtue of a foreign nuclear localization signal, it, like the murine Mx1 protein, blocks primary transcription (47), indicating that the Mx proteins of the two species act in a comparable way by recognizing the same or similar viral target structures.
Influenza A virus strains differ markedly in their virulence for a given host. It is conceivable that variations in Mx sensitivity could partly explain these differences. We therefore compared the sensitivities of different influenza A virus strains to inhibition by mouse Mx1 and human MxA proteins. We found that all influenza A virus strains tested were Mx1 sensitive, albeit to different degrees. In general, avian influenza viruses were found to be better inhibited than human strains. When tested in a minireplicon system, the highly pathogenic avian H5N1 strain A/Vietnam/1203/04 was more sensitive to the inhibitory action of Mx1 than the reconstructed 1918 H1N1 "Spanish flu" virus. Moreover, the human 1918 virus-based minireplicon system was virtually insensitive to inhibition by human MxA, in stark contrast to the avian H5N1 strain-derived system. Finally, we could show that the viral nucleoprotein (NP) is an important determinant of Mx1 sensitivity and may therefore represent the viral target structure.
(This work was conducted by Jan Dittmann in partial fulfillment of the requirements for an M.D. degree from the Medical Faculty of the University of Freiburg, Freiburg, Germany.)
Cells and viruses.Vero cells stably transfected with an Mx1 expression plasmid (Vero-Mx1) or a control plasmid (Vero control), as well as the mouse 3T3 cell line expressing Mx1 (3T3-Mx1) or not expressing Mx1 (3T3 control), have been described previously (4, 31). Mouse embryonic fibroblasts (MEF) were prepared as described in reference 21. MEF Vero, 3T3, 293T, and MDCK cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum and antibiotics. For Mx1 induction, MEF were treated with 1,000 U/ml of human hybrid IFN-α B/D, which is active on mouse cells (14).
A mammalian-cell-adapted variant of influenza A virus strain A/Fowl/Dobson/27 (H7N7), called FPV-B (17), was grown on Vero cells to a titer of 6 × 107 PFU/ml. Influenza A virus strain A/Udorn/307/72 (H3N2) (18) was also propagated on Vero cells (4 × 106 PFU/ml). Strain A/WSN/33 (H1N1) was grown on MDCK cells (6 × 107 PFU/ml). Strains SC35 (H7N7) (23) (3 × 108 PFU/ml; provided by Juergen Stech, University of Marburg, Marburg, Germany), A/Texas/36/91 (H1N1) (5 × 107 PFU/ml) (1), A/Panama/2007/99 (H3N2) (2 × 107 PFU/ml), and A/Wyoming/3/03 (H3N2) (2 × 107 PFU/ml; kindly provided by Terrence M. Tumpey, Centers for Disease Control and Prevention, Atlanta, GA) were grown in 8-day-old embryonated chicken eggs. Titers were determined by plaque assays on MDCK cells.
Antibodies and plasmids.Monoclonal antibody M143 was used to detect the Mx proteins (3), and a polyclonal rabbit serum was used to detect the influenza A virus nucleoprotein by Western blotting and immunofluorescence analysis. The monoclonal antibody against β-tubulin was purchased from Sigma (Munich, Germany).
Full-length cDNAs for the open reading frames encoding proteins PA, PB1, PB2, and NP of influenza virus A/Vietnam/1203/04 (H5N1) were amplified by PCR from viral RNA expression plasmids pPol1VN1203 PB2, pPol1VN1203 PB1, pPol1VN1203 PA, and pPol1VN1203 NP (30) and were cloned into pCAGGS (28) using Not1 and Xho1 restriction sites for PB2, PB1, and NP and Not1 and Nhe1 sites for PA. Expression plasmids pCAGGS-PB1, -PB2, -PA, and -NP derived from influenza A virus strain A/WSN/33 (2), as well as pCAGGS-PB1, -PB2, -PA, and -NP derived from strain A/BM/1/18 (43) (kindly provided by Chris Basler, Mount Sinai School of Medicine, New York, NY), have been described previously. Plasmids pDZ-PB1, pDZ-PB2, pDZ-PA, and pDZ-NP, derived from strain A/Texas/36/91 (43), were a kind gift from Dimitriy Zamarin and Peter Palese, Mount Sinai School of Medicine, New York, NY. Plasmids pHW2000-PB1, pHW2000-PB2, pHW2000-PA, and pHW2000-NP of strain SC35 (6) were obtained from Juergen Stech and Hans-Dieter Klenk, University of Marburg, Marburg, Germany, and plasmids pPolI/II-PB1, -PB2(E627K), -PA, and -NP, derived from strain A/Turkey/England/91, were kindly provided by Wendy Barclay, Imperial College London, London, United Kingdom (15).
The pPOLI-Luc-RT reporter construct, encoding firefly luciferase in the negative-sense orientation flanked by the noncoding regions of segment 8 of strain A/WSN/33, has been described recently (38). The second reporter construct, pPOLI-SP-Luc-RT, coding for firefly luciferase in the negative-sense orientation flanked by modified noncoding regions of segment 4 of strain A/WSN/33, as described by Neumann and Hobom for pHL1104 (26), was constructed by a PCR-mediated approach using plasmid pGL3-FF-Luc (Promega, Madison, WI) as a template and primers 5′-GACACGTCTCGTATTAGTAGAAACAAGGGTGTTTTTTCTTACACGGCGATCTTTCCGCC-3′ and 5′-GACACGTCTCCGGGAGTAGAAACAGGGGAAAATAAAAACAACCATGGAAGACGCCAAAAACATAAAG-3′. The resulting PCR product and the pHH21 vector were digested with the restriction enzyme Esp3I (Fermentas, St. Leon-Rot, Germany), and subsequently the insert was ligated into the vector. Expression plasmids pcDNA3-Mx1 and pcDNA3-MxA and the plasmids encoding the inactive mutants, pcDNA3-Mx1(K49A) and pcDNA3-MxA(T103A), have been described previously (38, 39).
Tissue culture infection experiments.Cells were incubated with the appropriate dilution of virus stock in phosphate-buffered saline with 0.3% bovine serum albumin for 1 h. Subsequently, the inoculum was washed off, and cells were incubated with DMEM containing 10% fetal calf serum. At the time points indicated, cells were incubated with lysis buffer (50 mM Tris [pH 7.5], 250 mM NaCl, 20% glycerol, 0.5% NP-40, 2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 2 U/ml benzonase, protease inhibitor [Complete Mini; Roche Diagnostics, Mannheim, Germany]) for 10 min on ice. The lysates were centrifuged for 1 min at 13,000 rpm, and the supernatants were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis.
For immunofluorescence analysis, the cells were seeded onto glass coverslips and infected with 2 PFU per cell. At 8 h postinfection (p.i.), the cells were fixed with 3% paraformaldehyde and permeabilized with 0.5% Triton X-100. The cells were then stained for Mx1 and viral nucleoprotein by using specific antibodies and fluorophore (Cy2, Cy3)-conjugated secondary donkey antibodies (Dianova, Hamburg, Germany). The cells were analyzed with a Leica TCSSP2 confocal laser scanning microscope.
For plaque assays, cells were incubated with serial dilutions of virus in phosphate-buffered saline containing 0.3% bovine serum albumin for 90 min. After removal of the inoculum, cells were overlaid with DMEM containing 0.6% agar (Oxoid Ltd., Basingstoke, Hampshire, England) and trypsin (1 μg/ml). Two to 3 days later, cells were fixed with 3.7% formaldehyde and stained with 1% crystal violet in 20% ethanol. Virus titers are expressed as PFU.
Influenza A virus minireplicon system.Transfection assays were carried out with human embryonic kidney cells (HEK 293T cells) seeded into 6-well plates. Cells were transfected using Lipofectamine transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. The indicated amounts of the three plasmids encoding the subunits of the viral RNA polymerase and the plasmid coding for the NP were cotransfected with 0.05 μg of plasmid pPOLI-Luc-RT or 0.5 μg of pPOLI-SP-Luc-RT carrying the firefly luciferase reporter gene. To provide a measure of transfection efficiency, 0.1 μg of the Renilla luciferase-encoding plasmid pRL-SV40-Rluc (Promega, Madison, WI) was cotransfected. To analyze the antiviral potential of Mx, increasing amounts of the Mx-encoding plasmids were cotransfected. As a positive control, plasmids encoding the antivirally inactive Mx1(K49A) and MxA(T103A) mutants were used. The negative control lacked the plasmid encoding NP. Equal amounts of DNA in the transfection mixtures were achieved by adding an empty pcDNA3 vector. Cells were harvested and lysed 24 h posttransfection. Firefly and Renilla luciferase activities were determined using the Dual Luciferase assay (Promega, Madison, WI) according to the manufacturer's instructions. Luminescence intensities were measured with a luminometer for 10 s each.
Influenza A virus strains differ in their sensitivity to Mx1.Initially, we compared influenza A virus strain FPV-B, a mammalian cell culture-adapted variant of the avian isolate A/Fowl/Dobson/27 (17), and the human isolate A/Udorn/72 with regard to their sensitivities to the antiviral effect of Mx1. For this purpose, we performed plaque assays on Vero cells constitutively expressing the mouse Mx1 gene. FPV-B was completely inhibited in Vero-Mx1 cells compared to Vero control cells (Fig. 1, upper panel), as expected from previous studies (37). However, much to our surprise, isolate A/Udorn/72 was able to form plaques on Mx1-expressing cells (Fig. 1, lower panel), although the number and size of plaques were lower than those in control cells (Fig. 1, lower panel).
We then extended our studies to a wider range of virus strains. Because viral replication does not necessarily lead to plaque formation and because most influenza A virus strains do not form plaques on Vero and mouse 3T3 cell lines, we determined the level of accumulation of the viral NP as a measure of virus growth in constitutively Mx1-expressing 3T3 cells by using Western blot analysis. Comparison of Mx1 expression levels in 3T3-Mx1 cells with those in IFN-treated primary MEF derived from congenic B6.A2G-Mx1 mice (21) revealed comparable amounts of Mx1 protein accumulation in the two cell cultures (Fig. 2B). In addition to strains FPV-B and A/Udorn/72, we used SC35 (an influenza virus strain that was derived from A/Seal/Massachussetts/1/80 by serial passages in chicken embryo cells and is pathogenic for chickens [23]) as well as strains A/WSN/33, A/Texas/36/91, A/Panama/2007/99, and A/Wyoming/3/03, which are derived from human isolates. At 6 h p.i., 3T3-Mx1 and 3T3 control cells were lysed, and Western blot analysis using a polyclonal rabbit serum against the viral NP was performed. Analysis of the viral NP revealed differences in signal intensities between the various strains (Fig. 2A). The intensities of the NP signals were strongly reduced in Mx1-expressing cells from those in control cells for the avian strains FPV-B and SC35 and for A/WSN/33, whereas only slight reductions in intensity were observed for the four human strains A/Texas/36/91, A/Udorn/72, A/Panama/2007/99, and A/Wyoming/3/03 (Fig. 2A). In order to exclude the possibility that the expression levels or the nuclear localization of the Mx1 protein was affected by infection with these less-sensitive viruses, we determined the levels of Mx1 by Western blotting and the subcellular localization of Mx1 by immunofluorescence analysis. Figure 2B shows that infection of 3T3-Mx1 cells by strain A/WSN/33, A/Texas/36/91, or SC35 did not alter Mx1 protein levels in the cells. Parallel detection of β-tubulin confirmed the loading of equal amounts of cell lysates. In addition, the nuclear localization of Mx1 was not affected by infection with the viruses (Fig. 2C and data not shown).
Inhibition of influenza A virus minireplicon systems derived from different strains by Mx1.Previous publications have shown that Mx1 affects primary transcription of influenza A virus (22, 31). Therefore, we reasoned that the effect of Mx1 should be visible in minireplicon systems derived from influenza A viruses. It was shown previously that expression of reporter gene activity from a minigenome containing the noncoding regions of influenza A virus segments quantitatively reflects the activity of the viral polymerase complex in virus-infected as well as cDNA-transfected cells (24). To investigate differences in sensitivity to Mx1, we used minireplicon systems derived from strains A/WSN/33, A/Texas/36/91, SC35, and A/Turkey/England/91. First, we defined settings that allowed us to directly compare the viral polymerase activities of the different systems. For this purpose, we titrated the amounts of the polymerase- and NP-encoding plasmids to determine the conditions for each system that lead to 50% of the system's maximum activity (data not shown). We then used these settings to analyze the inhibitory effect of Mx1. By cotransfection of increasing amounts of an Mx1-encoding plasmid, we determined the amount of Mx1 plasmid necessary for 50% inhibition. The resulting curves are shown for the A/WSN/33 and A/Texas/36/91 systems (Fig. 3A). The largest amount of plasmid used for Mx1 (750 ng) was also used for an expression plasmid encoding the inactive mutant Mx1(K49A) (32). As expected, Mx1(K49A) did not have an inhibitory effect, and the respective luciferase activities were set to 100%. For both A/WSN/33 and A/Texas/36/91, increasing amounts of the Mx1-encoding plasmid led to decreasing luciferase activities. However, the expression of the reporter gene was much more sensitive to Mx1 in the system derived from strain A/WSN/33 than in that derived from A/Texas/36/91 (Fig. 3A).
The amount of Mx1 plasmid needed for 50% inhibition was then fine-titrated within the range determined in the first series of transfections (10 to 100 ng of Mx1-encoding plasmid for A/WSN/33 and 300 to 400 ng for A/Texas/36/91). The exact amounts were determined to be 37.5 ng for the A/WSN/33 system and 375 ng for the A/Texas/36/91 system (Fig. 3B).
Using this strategy, we also determined the amount of Mx1-encoding plasmid necessary to inhibit two avian systems derived from strains SC35 and A/Turkey/England/91, respectively. Because the assay did not work efficiently with the conventional reporter construct, pPOLI.Luc-RT, which is based on the wild-type noncoding regions of segment 8, we used pPOLI-SP-Luc-RT, a reporter construct with enhanced expression properties based on the modified noncoding regions of segment 4 of A/WSN/33, which has been described by Neumann and Hobom (26). To enable comparison of the results with those of our previous assays, we also included the A/WSN/33 expression plasmids in this setting. We titrated plasmid amounts as described above and found that 200 ng of the Mx1-encoding plasmid was necessary to reduce the activity of the system derived from A/WSN/33 to 50% relative to the activity observed when the mutant promoter construct was used (Fig. 3C). In contrast, cotransfection with only 5 or 6 ng of Mx1-encoding plasmid resulted in 50% inhibition of the polymerase activities of SC35 and A/Turkey/England/91, respectively (Fig. 3C). These data clearly demonstrate that there are large differences in sensitivity to Mx1 between the strains. The avian strains A/Turkey/England/91 and SC35 are the most Mx1 sensitive. The human strain A/Texas/36/91 is the most resistant, whereas A/WSN/33 displays an intermediate phenotype.
The viral nucleoprotein as a possible target of Mx1 action.Next, we used the minireplicon system to identify the Mx1 target protein. For this purpose, we exchanged expression plasmids between the different strains to determine whether an Mx1-sensitive minireplicon system could be converted into a more resistant system and vice versa. Unfortunately, the exchange of plasmids encoding PB1, PB2, or PA led to a strong decrease in the activities of the mixed polymerase complexes, making a comparison with the original minireplicon systems very difficult. However, exchanging the NP-encoding plasmids was compatible for the systems derived from strains A/WSN/33, A/Texas/36/91, and SC35, resulting in approximately the same luciferase activities as those measured with the original systems. We used the minireplicon systems at 50% of maximum activity and cotransfected the amount of Mx1-encoding plasmid leading to 50% inhibition. The inactive mutant Mx1(K49A) was used as a control, and activity with this mutant was set to 100%. In this context, the effect of exchanging the NP-encoding plasmid was determined. We observed approximately 50% inhibition when we used 37.5 ng of the Mx1-encoding plasmid and the NP from strain A/WSN/33 in the A/WSN/33 system, as expected (Fig. 4A). When we used the same amount of the NP-encoding plasmid of A/Texas/36/91 in the A/WSN/33 system, luciferase activity in the presence of Mx1 increased to 70% of that in the presence of Mx1(K49A). In contrast, the use of the NP of SC35 resulted in a reduction of luciferase activity to 6% by Mx1 (Fig. 4A). For the system derived from strain A/Texas/36/91, the use of the original NP resulted in an inhibition of approximately 50% by transfection of 375 ng of the Mx1 expression plasmid (Fig. 4B). Use of the NP of A/WSN/33 or SC35 strongly increased the inhibitory potential of Mx1, leading to a reduction of polymerase activity to 13% or 3%, respectively (Fig. 4B). When we used the avian system derived from SC35, approximately 50% inhibition of polymerase activity was achieved by cotransfection of 6 ng of the Mx1 expression plasmid. Exchange of the NP resulted in a partial offset of inhibition by Mx1, bringing the level of inhibition to only about 25% with the NP-encoding plasmid of A/WSN/33 or A/Texas/36/91 (Fig. 4C). Taken together, these data reveal that NP has a great impact on the sensitivity of the minireplicon system to Mx1, since it was possible to convert a sensitive minireplicon system into a more resistant system and vice versa by changing the NP-encoding plasmid. Therefore, our approach identified the influenza A virus nucleoprotein as a possible target structure of Mx1.
Antiviral potential of mouse Mx1 and human MxA against highly virulent 2004 H5N1 and 1918 H1N1 strains.So far, our results have shown that although there were differences between the influenza A virus strains, all were sensitive to the antiviral action of Mx1. We therefore considered whether highly virulent strains, such as the pandemic virus of 1918, A/BM/1/18 (43), or a recent H5N1 isolate from a human patient with a fatal case, A/Vietnam/1203/04 (25), are also sensitive to Mx1 action. To answer this question, we transfected 293T cells with the appropriate expression plasmids for minireplicon assays. We titrated the plasmid amounts to achieve 50% of maximum activity: 25 ng of each polymerase-encoding plasmid of A/BM/1/18 or 150 ng for A/Vietnam/1203/04. Using these conditions, we cotransfected increasing amounts of the Mx1-encoding plasmid or a plasmid encoding the inactive mutant Mx1(K49A) and determined the amount of plasmid needed for 50% inhibition (Fig. 5A). Exact titration of the amount of Mx1 expression plasmid required for 50% inhibition revealed it to be 125 ng for the system derived from A/BM/1/18 and 25 ng for the A/Vietnam/1203/04-derived system (Fig. 5B). This suggests that the sensitivities of the highly pathogenic viruses follow the same pattern as those of their moderately pathogenic counterparts.
Since both viruses were isolated from humans with fatal influenza A virus infections, we also tested the effect of human MxA on the minireplicon system. We used an MxA-encoding plasmid or a plasmid coding for the inactive mutant MxA(T103A) (33) and performed titration as described above. We found that MxA coexpression had a strong inhibitory effect on the avian system and that only 110 ng of the MxA-encoding plasmid was needed for 50% inhibition of the A/Vietnam/1203/04 system (Fig. 5C). However, with 1,500 ng of the MxA-encoding plasmid, no inhibitory effect on the polymerase activity of A/BM/1/18 was detectable (Fig. 5C). We also studied the sensitivity of the polymerase of A/Texas/36/91 to MxA expression and determined a similar lack of inhibition by 1,500 ng of the MxA-encoding expression plasmid (data not shown), suggesting an unexpected insensitivity of the polymerase complex of human influenza viruses to the expression of MxA.
Here we demonstrate that influenza A virus strains differ markedly in their sensitivities to the antiviral action of Mx proteins. The amounts of Mx needed to inhibit different influenza virus replicon systems differed greatly. We further show that the viral NP is a determinant of viral sensitivity to Mx. NP is a major component of the viral ribonucleoprotein complex and is essential for viral transcription and replication. Our findings indicate that NP is a target of Mx.
We found that influenza A virus strain FPV-B was highly susceptible to Mx1, confirming earlier studies (37). By screening a panel of influenza A virus strains, we could identify viruses that were able to replicate reasonably well in the presence of Mx1. Interestingly, influenza A viruses with heightened Mx resistance represented human isolates, whereas the more sensitive viruses were of avian origin. Importantly, virus sensitivities were reflected in the Mx sensitivities of minireplicon systems. Thus, the minireplicon system based on the avian H5N1 virus currently circulating in Asia (25) was sensitive to the antiviral action of Mx1. It is therefore tempting to speculate that avian influenza A viruses have only limited opportunity to adapt to the mammalian host, partly because of the mammalian Mx system. Successful human strains were presumably selected for low sensitivity to mammalian Mx proteins during the course of their adaptation. The intermediate sensitivity of strain A/WSN/33 was remarkable and is not easily explained. The virus was extensively passaged in laboratory mice that are negative for a functional Mx1 gene (36) and may have lost some Mx resistance. Further studies analyzing primary human and avian isolates with respect to their sensitivities to mammalian Mx proteins are under way to test this hypothesis.
Interestingly, we found that minireplicons based on the pandemic strain A/BM/1/1918 as well as minireplicons of strain A/Vietnam/1203/04 (isolated from a human patient with a fatal case) were inhibited by Mx1. These results fit very well with recent in vivo experiments showing that Mx1+/+ mice survive infection by both viruses with doses that are lethal for genetically defective Mx−/− mice (34, 44). Nevertheless, the polymerase activity of A/BM/1/1918 was inhibited less strongly by mouse Mx1 than that of A/Vietnam/1203/04. The difference in sensitivity between the two strains was even more pronounced with human MxA. MxA inhibited the polymerase activity of A/Vietnam/1203/04 more efficiently than those of A/BM/1/1918 and A/Texas/36/91, which were both virtually insensitive. It is therefore likely that human MxA provides an efficient block to zoonotic transmission of avian influenza A viruses, including H5N1 viruses that actually circulate in Southeast Asia. Successful viruses that adapt to and spread from humans to humans may have evolved a relative insensitivity to the antiviral activity of MxA.
Furthermore, there may be an inverse relationship between the capacity of viruses to antagonize IFN production and their sensitivity to Mx. It was shown by Hayman et al. that viruses that are less potent at inhibiting IFN induction are more resistant to IFN treatment than viruses that are strong suppressors of IFN (12).
The strain-specific differences in Mx sensitivity were used to analyze the viral target structure of Mx1. By exchange of the NP-encoding plasmids, we could convert a sensitive polymerase complex into a more resistant one, and vice versa. This shows that NP is an important viral determinant of Mx1 sensitivity and therefore a possible target structure of Mx proteins. This role of NP is not without precedent: Turan et al. were able to show an interaction of NP with a variant of human MxA carrying a foreign nuclear localization signal (45). Furthermore, MxA interacts with the NP of Thogoto virus, a member of the orthomyxovirus family (20, 46). In addition, the PB2 subunit of the influenza virus polymerase complex has been proposed as a target structure of mouse Mx1 (16, 41). It is therefore conceivable that the viral ribonucleoprotein complex, consisting of genomic RNA, NP, and viral polymerase, is the target structure of Mx proteins.
In summary, our results show that there are large differences in the sensitivities of influenza A virus strains to inhibition by the IFN-induced Mx proteins. The avian influenza virus strains are very sensitive, whereas strains of human origin are more resistant. Most remarkably, the polymerase complex of the 1918 pandemic influenza A virus was not affected by the highest MxA expression levels used in our test system, suggesting a remarkable adaptation to human cells. The viral NP proved to be a determinant of Mx sensitivity, supporting the hypothesis that viral ribonucleoprotein complexes are target structures of Mx proteins.
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ko1579/1-8) and the German-Israeli Research Foundation (GIF-841/04) to G.K. and O.H. and by grants from the National Institutes of Health (R01AI46954 and P01AI58113) to A.G.-S.
We thank Peter Staeheli and Friedemann Weber for discussions and critical comments on the manuscript. We are grateful to Wendy Barclay for A/Turkey/England/91 expression plasmids, to Jürgen Stech and Hans-Dieter Klenk for virus isolates and SC35 expression plasmids, to Christopher Basler for 1918 expression plasmids, to Terrence M. Tumpey for influenza virus strains A/Panama/2007/99 and A/Wyoming/3/03, and to Dimitriy Zamarin and Peter Palese for virus isolates and A/Texas/36/91 expression plasmids.
- Received 10 August 2007.
- Accepted 24 December 2007.
- ↵*Corresponding author. Mailing address: Department of Virology, University of Freiburg, Hermann-Herder-Strasse 11, 79107 Freiburg, Germany. Phone: 49-761-2036623. Fax: 49-761-2036562. E-mail: georg.kochs{at}uniklinik-freiburg.de
↵† J.D. and S.S. contributed equally to this work.
↵‡ Present address: Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029.
↵▿ Published ahead of print on 16 January 2008.
- American Society for Microbiology


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Despite unsettled market conditions, a foursome of biotechs priced their IPOs within their proposed ranges ahead of first-day trading Thursday and helped push the year-to-date tally of money raised in public market debuts well ahead of last year's pace. Coming during a week when escalating trade tensions between the U.S. and China have propelled indexes downward, the offerings will be closely watched by the bolus of companies in the IPO queue.
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Itacaré > Real estate > Land and houses for sale
Vende-se casa
Rua do Canoeiro - Km 04
House for sale. The lot is 1.500m² and located 4km from the town. The house itself is 80m² with an extra 40m² of balcony area. The house is equipped with a full kitchen and island, bathroom and spacious living room that opens up to the balcony. Comfortable upstairs bedroom, with a balcony that looks out into the valley.
Contact: Ana Laura
☎ +55 73 - 99849 2134
☎ +55 73 - 99849 2134


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Located in the heart of historic downtown Blacksburg, Main Street Inn is within walking distance to Virginia Tech. Guests can enjoy dining, music theatre and shopping...all a short stroll away.
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Main Street Inn
Welcome to Main Street Inn
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More Lodging
For more lodging options outside of the downtown area please visit the Montgomery County Tourism website.
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text: This Friday we will get the always important monthly jobs report. We will cover the report after its release in Real Money, but investing is all about looking forward, so here's my take on what we might see and how markets will react. Nothing can stop a rate hike in September (and probably December too) While there will be plenty to digest in the jobs report, investors should assume that baring some really wild disaster, rate hikes in September and December are just about a given. There's lots of potential for noise in the employment report, so anything can happen in any given month. However there is too much momentum in the economy now to think that hiring will suddenly dry up. The Fed knows that, so even if we were to get a quirky -50,000 jobs report or some such, they will have plenty of confidence in the overall strength of the labor market to keep hiking. The Powell doctrine: gradualism There's much talk about the Powell doctrine after the Chair's speech at Jackson Hole. You can see my thoughts on the speech itself here. Powell wants to raise rates slowly but steadily. They want to remove policy accomodation and move toward neutral (and probably a bit beyond neutral), which should prevent any inflation pressure from getting too strong. However, they want to move slowly enough that it would allow them to see any economic damage incurred before it became too severe. Here's the thing to keep in mind: pausing will make them nervous. In the speech Powell made an analogy that if you are unsure of the strength of a medication, you start with a small dose. To carry that analogy one step further, if you are giving small doses and you don't see any change in the patient, you need to dose again. Hence, if they hike in September and unemployment is still falling, they hike again in December. If unemployment is still falling, they hike again in March, and so on and so on. So these next 3-4 jobs reports are all about set up for 2019 If anything, it looks to me like the pace of hiring is accelerating. Both the rolling 6-month and 12-month averages for non-farm payroll growth are sloped upward. The 6-month average is currently +221,000 after spending most of 2017 in the +180,000 area. We need to add only around 120,000 to keep unemployment steady, so if we keep up a pace north of 200,000, unemployment ought to fall rapidly. Supporting data all suggests hiring should keep up a very strong pace: business confidence surveys are high with hiring intentions specifically jumping higher in recent months. As I said above, the employment report can be noisy, so I wouldn't be shocked by a weaker report, but your base case should be around 200,000/mo for at least the next 3-4 months. And if that happens, the Fed is not only hiking in September and December, but also in March and probably June. Could they hike even faster? The bar is quite high for them to hike faster than once per quarter. It would take sustained Core PCE inflation prints meaningfully above 2%, i.e., the 2.3-2.5% range. As long as inflation isn't obviously accelerating, Powell's gradualism doctrine will rule the day. This is not my base case. I'm pretty convinced that the secular forces holding inflation down plus gradual hikes will be enough to keep rates under control. That said, I see much higher odds that they'd be forced into hiking faster than take an extended pause... What about a pause in early 2019? It seems to be the consensus that the Fed will pause in either March or June next year. Indeed the market seems to be pricing that the Fed will stop hiking in mid-2019. While I think it is possible that the Fed will skip on their quarterly hike in either 1Q or 2Q 2019, I'm betting the other way. Its one thing to assume in the hypothetical that a pause will make sense. It is another thing to make that decision in the moment. We are hearing some regional presidents saying that they want to pause once the Fed gets to neutral policy, but how would you know you are there? I think when that moment comes, if the economy is still ripping, those calling for a pause will be in the minority. In other words, if the economy is ripping, policy obviously isn't neutral! I continue to believe that the Fed will keep hiking until they've done damage to the economy. Trade ideas There are three ideas I've been suggesting based on the view that the Fed hikes more times in 2018-2019 than is currently priced in: Short 3-7 year Treasuries. This is the part of the yield curve most vulnerable to additional rate hikes. I prefer to do this as a pair trade with... Long longer-term bonds. The only way the curve re-steepens is if inflation pressure builds, which as I said above the Fed isn't actually going to allow. I like owning 10+ year bonds, and particularly like a pair trade of long 20-30 year and short 5-year. Long USD. With DXY coming off over-bought condition, it is a nice buy here.


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Guitarists true to one guitar
By Miguel Ángel Ariza
Most of our idols, having lived at a time when music was a gigantic multi-millionaire business, ended up very rich, and some of us can ponder that having all that cash, allowing them access to the finest guitars ever made, each of their collections would grow too big to fit in their mansions, right?
Well many of them did just that, but curiously certain players have stuck with the same specific model of guitar, ignoring all the beauties around them, to which they had total access, and forge their image almost seen as incomplete, if not for the "his" model draped over the shoulder. We're talking about guitarists like Slash or Paul Kossoff, and their Gibson Les Paul Standard; just like Angus Young, Tony Iommi, and Derek Trucks with their Gibson SG, we could also throw into the lot people such as B.B. King and Chuck Berry with their Gibson hollow bodies, either the 335 or the 345. Can you imagine either of the last two taking the stage with a Fender Stratocaster? It wouldn't be the same artist, would it?, that's why we pay tribute in this article to guitarists true to one guitar.
First of all we must point out that it isn't the same to always play the same model as to play the same guitar. We know that there were many Lucilles; we also know that the Fender Stratocaster is what it is today thanks to a fellow called Jimi Hendrix, but let's not forget the burnings, breakings, and throwing many of them into the air at gigs, so those two geniuses are good examples of artists true to a model but not a guitar specifically. Something much less promiscuous happens when players attach themselves to a few guitars, even choosing one over the others, like Stevie Ray Vaughan and his '59 Strat, Brian Setzer and his different Gretsch 6120s, the original being from '59, his favourite, and with which he played almost all his time while in the Stray Cats, or Danny Gatton and the 2 or 3 Fender Telecasters that he played for a very long time (the most iconic being the '53, and longest used).
Still, if we can further narrow the relationship between player and a single guitar, we have to mention people like Joe Strummer for instance, whose Fender Telecaster, although it wasn't the only one, was with him since the mid-70s until he left us in the 21st century; or Bonnie Raitt with her unpainted Fender Stratocaster whose body dates back to '65, the only guitar she has played in every show since she got it in 1969. Another guitar that might also belong in this group is Bob Marley's Gibson Les Paul Special, his main guitar since the beginning of his career until his death and the reason why his signature sound is always linked to reggae music.
And finally, knowing that there are many love stories between players and models that we have missed, we must name some that seem the most tender relationships to us, be it for how long, emotional or idyllic the union was . The first is the guitar made by Hermann Hauser for Andrés Segovia who for many was the best guitarist on Earth and who played that guitar between 1937 until 1962. But let's go to the electric world, where we don't have to go back so many years to find love like that. Starting with the guitar that a young fellow mad about the 6-string and his father designed and built together in their garage, giving birth to what we know today as Red Special and which has always been linked to that teen: Brian May. While it's true that he sometimes plays a replica, it's the original guitar which defined the sound of Queen, and of an era.
A relationship similar to what May has with his Red Special is the one Mr. Bruce Springsteen had with his hybrid between a Fender Esquire and Telecaster, until a few years ago his luthier advised him to retire it from touring; there isn't an image of the Boss without a Telecaster, it has been his companion for more than 4 decades. It's no wonder that with so many tours and over 3-hour concerts his luthier has convinced him to use another guitar. However, this retiring early thing is not easy to pull off, just ask Neil Young who is still shredding his legendary "Old Black" (a Gibson Les Paul Goldtop from '53, somewhat modified) in everyone of his concerts where he has to get his fans pumped up. An absolute delight to see this guitar still in action and in top form.
We'll take the last step before we close by mentioning two of the absolute geniuses of the electric guitar, and both share having tailored his sound and style with one guitar. We're talking about the most-adored Rory Gallagher and Peter Green. It is true that Peter Green went on with his career beyond Fleetwood Mac with other guitars, but it is clear that he is and will be remembered for what he did in those early years in the Brit band with 'Greeny' over his shoulder, which for some is the best sound ever to come off a Les Paul (to be more exact, a '59 Gibson Les Paul Standard, and as we have noted many times in Guitars Exchange, it later belonged to Gary Moore, and nowadays it can be seen in Metallica tours with Kirk Hammett).
Rory Gallagher meanwhile, was never apart from his Fender Stratocaster (the first to reach Ireland by the way) since meeting her in the 60s until the day of his death. His story and that of his '61 Strat with such characteristic relic are as linked together as fabled King Arthur and Excalibur. It's not one without the other.
And to top off this piece of love between players and guitars, we have to do it with the man who said many years ago that "the day Trigger goes, so do I", this bond between this Martin N-20 baptised as Trigger in 1969 and Willie Nelson goes beyond human love. It is known that his audiences hold their breath, and treat it as if it were church liturgy when one of the crew place Trigger in the middle of the stage before the show starts. It's this complete connection between the guitar and artist that makes these inanimate objects take on a life of their own in the minds of his fans, who commend all kinds of skills and abilities to them, which though it may seem a biblical tale, they may well possess, as many of the folk we have mentioned here have given themselves to one or very few guitars, given the abundance of models and guitars that exist, and how easy it would be for them to get them, is an authentic miracle.


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Look Out (Western Girl Series No. 4)
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Western Girl Series 4
When the gun-glint matches the glint of her eye - Oh, my, Oh, my! It's a good time, stranger, to say good bye. Good bye! Good bye! - Walter Juan Davis "A room without pictures is like a room without windows." - Ruskin We are showcasing a nice line of Framed and Unframed Pictures at popular prices. We also make picture frames Midgley-Bodel Co., 33 E. First South Street


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I am going to, but I am thinking about it like this. I know that a core, without the end I have added, works, as you can see in the bedini patent. So there is no point in testing what has been tested. I am going to make a core with the end like mine and try that but no matter what happens I am building the big one. 1 because this is my hobby so I don't mind spending 200 every few months to try something new 2. I have been running everything through my head for about a year now for THIS machine I have had the idea that long but took a year to think about it. So I am fairly certain that all my research on coil windings and the concepts in this machine will pay off in some way. 3. Basically I want to see what happens when I build the whole thing...if I got absolutely nothing out of the coils I would still build it. To see if I can make changes because I believe this will work. Other things I have thought were cool ideas but this one just clicks for me.
walter russell says everything is a cycle every action causes a reaction that in turn causes the action again in a cycle. noone up until this idea (unless i missed one somewhere) has put that thinking into magnetic loops. every time a magnetic loop is broken in this machine it creates another magnetic loop wich when broken cause the first magnetic loop again. So action reaction action. along with the action of the incoming magnet canceling the drag of the outgoing magnet would cause (if it works) the machine to spin as though there were no magnetic locking or "coging" but it would actually lock very strongly . But the drive would never see it because of the equalization in drag and pull forces.


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Last Updated on
Expedition Safety Concerns and Tips
Alcohol is the #1 most dangerous variable on many GC river trips. Folks should be as aware of this as they are about their PFD. Alcohol is the #1 leading cause of death and injury on river trips. Inebriation leads to poor coordination, poor body function( congestive heart failure, unbalanced electrolytes, low immunity, poor circulation, etc) and Judgement that can inversely affect the group in a big way. If there will be alcohol on your expedition, talk about what responsible drinking is before the trip.
Poor Health can impact a group inversely. The Grand Canyon is a harsh environment. Intense heat, freezing temperatures, swimming in cold water temperatures. Possibilities of fractures, cuts, open sores, foot rot, sprained ankles, sunburn, hyponatremia, dehydration, drowning, hypothermia, infection, septic, the list is endless. If an individual group member is not healthy going into the trip, that individual will no doubt be very unhealthy throughout the trip. A "Do It Yourself" trip is a working vacation. If any one possible member of the group is not a functioning "Do It Yourselfer", or HEALTHY, DO NOT invite them. They will not have fun, and the liability will ruin everyone's trip. Mental health should also be taken into consideration. Extreme personalities will amplify on an expedition.
Medical Experience is a must! Starting from First Aid, anatomy, advanced First Aid, WFR, EMT, Emergency Responder, RN, Doctor. A Good Trip Leader has some type of medical experience or training. Emergency Responders like firemen, paramedics are priceless members of an expedition.
Trip Leader. For any expedition, a group must be able to make intelligent decisions together. The Leader can facilitate the benefits of safety in numbers with a group knowledge collective. One example, a medical team. There may be an RN with more experience in medical issues. A trip leader may choose to appoint or lean on the RN for medical care. The Trip Leader should be abreast in group dynamics and conflict resolution. Also, the Trip Leader should have working knowledge of First Aid, boat repair, and boat extraction. In addition the Leader should know how to repair a boat and equipment vital to the safety of the group. The Trip Leader should be able to put the group above himself.
Expedition Members. Positive, communicative, group oriented, hardworking, willing to help. Many hands make light work. When a 16 person group all pitches in to load the boats it can seem like a well oiled machine and group work gets done fast and efficiently. When individuals are not helpful, and helpless a group can splinter into subsets that fracture the group dynamics and make leadership difficult. This can be particularly troublesome in an emergency. Every Members has strengths and weaknesses. A good group will work together to use each other strength and fill each other weaknesses. Be a Team Player!!
Groups of less than 8 people are at more of a disadvantage in many areas and should be particularly attentive to being more adept at the rigors and needs of an Expedition.
Bring Proper Clothing and Gear. Bring all needed safety gear and Leave No Trace camping.


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Grown in the heart of Germany - catering for your high-carb synthesis appetite. No more digital diets and virtual simulators, this is the real deal.. If you want to hear the engine roar, we have the tool on heavy fuel: the Waldorf Pulse.
Did we mention the filters? Their creation called for self-experiments of our engineers - some even under extreme daylight deprivation. It was in a night of thunder and lightning when eventually they came to life…
The Pulse + has built in Midi to CV converter for controlling other CV synths. it also has an audio input to the filter. The synth is fully midi controllable. 2u rack unit with a smooth high end sound.
info below from VintageSynthExplorer
The Waldorf Pulse is a monophonic synthesizer equipped with three oscillators and an arpeggiator. Its 100 patch programs are divided into 59 presets, 40 user sounds and one random sound. In addition to some standard modulations, four freely routable modulation units with selectable source, amount and destination give almost unlimited possibilities for analog bass and lead synth sounds.
On the Pulse+ model, there is a built-in MIDI to CV converter with CV/Gate input and output for communicating with your old analog gear. All parameter changes are sent and received as MIDI controller messages. There are up to 16 modulation sources, 4 routable modulation units with adjustable source, amount and destination parameters, 6 knobs and 4 buttons for convenient editing. This and a lot more puts the Waldorf Pulse a class of its own.
**Includes the synth itself and the PSU. Also includes original manual. no original box.
Click link below for manual


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Nativo's Next Gen Ad Server and Monetization Platform Gains Wide Adoption with Premium Publishers
MarTech Series | May 28, 2019 - Nativo, the leading monetization platform for premium publishers, continues to quickly expand its footprint with publishers looking to drive their bottom-line and explore alternative best-in-class solutions to Google Ad Manager. Nativo's platform now serves more than 500 major publishers across the US and UK, including The Globe and Mail, Tribune Publishing, Lonely Planet and The Philadelphia Inquirer. With Lonely Planet, Nativo's platform helped support sponsored content volume growth by as much as 300%.
Nativo's next generation ad server and monetization platform allows premium publishers to easily adopt new high-impact formats and accelerate speed to market with innovative advertising solutions. More specifically, Nativo's key ad serving capabilities bring publishers unique benefits:
"Our partnership with Nativo has enabled us to provide deeper and smarter metrics for our native advertising clients. We are seeing great success with this platform." - Robin Gruen, VP, Creative & Strategy at Studio 1847, a Tribune Publishing Company
"Nativo's ability to manage the complexities of branded content executions simultaneously across owned and operated sites as well as our Alliance publisher partners is a huge differentiator from alternative solutions in the market. Not only are we able to easily scale sponsored content ads across multiple Globe and Mail digital properties, but also seamlessly offer our clients the additional reach of our Globe Alliance partners." - Tracy Day, Managing Director, Creative Studio and Ad Innovation at The Globe and Mail
"Nativo clearly designed their ad server with publishers' unique business needs in mind. The platform's advanced capabilities have allowed us to execute branded content campaigns with full control and visibility. For example, their MOAT integration and ability to track advanced analytics such as scroll depth and velocity have given us more insightful data to drive successful results and increase satisfaction with our advertisers." - Lauren Amato, Director of Client Success at The Philadelphia Inquirer
Learn more about our unique offerings on MarTech Series.


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Gary Carlson
Anoka High Class of 1965
Personal Info
- Name:
- Gary Carlson
- Graduation Year:
- 1965
Register to view Current location, Gender, Birthday, Relationship Status, Children, Employer, Military, College, and Interests
Memories of Gary Carlson
What I'm doing now:Register to see what Gary Carlson is up to now!
Gary Carlson's Latest Activity:
|- Registered as a AHS Class of 1965 alumni. 2011


text:
Tagged By@madandrea ... This should be fun. Rules: every 3rd gif in your collection followed by these quotes in order (no skipping no cheating!) quotes: Parents Reaction when I was born. Me on my first day of school. When I learned to ride a bike. When I got a gift from my bff My feelings when I got my first crush Crush's reaction when I told him/her Getting the "Talk" Waiting for exam scores When I realize I failed. When I realized I actually did pass. Realizing I had a crush on my teacher. When i finally graduated! When I got my first good job. How I feel about the rest of my life. Alright! Have Fun!
My Parents When I was born: "Yeah, I totally created that."
me on my first day of school: Fun times at show n tell.
When I learned to ride a bike. (I'm the kid in the blue ) lol
When I got a gift from my bff. (I guess I was underwhelmed... )
my feelings when I got my first crush. (pretty accurate lol.)
My crush's reaction when I told him. ( also fairly accurate.)
Getting the TALK. LMBO YES.
Waiting for Exam Scores.
Realizing you failed. Lol
Realizing you actually passed. Bahahaha ummm...
Realizing I had a crush on a teacher. Ummmmm.... No? Lol
When I finally Graduated Lol happy prancing.
When I got my first good job. what was my first job.... Omg.
How I feel about the rest of my life. LMAO yes.
Final third gif. xD AWWWWWE ^^ tagging@VixenViVi@RihannaTiaMay@Vlargo@thePinkPrincess@Mercii@kpopandkimchi@moonchild03@EmilyGardner@Krin@BreeHubler@sugalessjams@reyestiny93@BapMokja@ElleHolley@SarangRavi@Torchix@TerraToyaSi@CreeTheOtaku@Poojas@SugaOnTop and whoever else wants to.


text: You could make a download version of the page that is an .hta (html application). I have made a few .htas for folks to use in my department. They are very handy and easy to do. (But work only on Windows.) This is copied from http://msdn.microsoft.com/workshop/author/hta/overview/htaoverview.asp . In the head tag you can include a path to the icon: <HEAD> <TITLE>My Monster Application</TITLE> <HTA:APPLICATION ID="oMyApp" APPLICATIONNAME="monster" BORDER="none" CAPTION="no" ICON="/graphics/creature.ico" SHOWINTASKBAR="no" SINGLEINSTANCE="yes" SYSMENU="no" WINDOWSTATE="maximize"> </HEAD> When you make a shortcut to the application that icon will be what shows on the desktop or wherever. I created a script in my .htas that will create a shortcut to the application. You can choose to place the shortcut on the desktop or in a number of other places. You would have to do some other scripting, of course, to maybe automate setting things up after its downloaded, and you'd probably have to make it downloaded as a zip file or something with instructions on how to use it. Not perfect, and probably not what you were looking for, but it would work. Somebody smarter could probably come up with something simpler/better. Diane P.S. Sorry if everthing I post comes out lame. You have no idea how hard it can be to type with a parrot on one hand and a chatty boyfriend making snide comments all the time. On Tuesday, June 22, 2004, at 02:40 PM, thelist-request at lists.evolt.org wrote: > Subject: [thelist] Creating a desktop link to a web page > Reply-To: Casey Crookston <casey at thecrookstons.com>, > "thelist at lists.evolt.org" <thelist at lists.evolt.org> > http://www.alistapart.com/discuss/dynatext/6/ > > We're all familiar with the "click here to add this page to your > favorites" > link. I have a client who would like to take this one step further. > > She's developed a web-based tool which has become popular with some of > her > clients, and would like a link on the web page, which, when clicked, > creates > a desktop icon. later, when that icon is double clicked, a browser is > opened > to her page. > > How would one go about doing this? I don't have any problem creating > the > icon file (.ico), but I don't know how to code the link to create the > icon > on the user's desktop. Is this even possible? > > Thanks! > > Ccrookston *** Don't be afraid to try something new. An amateur built the ark. Professionals built the Titanic. -unknown


text: Collin's shoot was the most epic Sr. shoot I have ever done. The original plan was to have the shoot only last one session. Collin wanted something extreme. I decided the most extreme and visually unique place I could think of was an abandoned missile silo. The silo I found was abandoned since the cold war era and is just sitting in the middle of nowhere. Due to the dust and mold we had to wear face masks in the silos...my mistake was not having Collin take off his mask in more photos. This proved in the end to be a problem...but I was more than happy to fix it. I had the pleasure to go on a second shoot with Collin. This second shoot was totally different but I was still in love with the final photos. -Devin


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"I have always strictly adhered to the Constitution of the Russian Federation. It is clearly stated in the Constitution: no more than two consecutive terms. Now I have a second term. As you remember, I was twice president before that, and then left the post of president, because the Constitution did not allow me to be elected for a third time, that's all. I intend to adhere to this rule in the future," Putin said in response.
At a meeting with the chief executives and editors-in-chief of international news agencies, a female participant, asking her question, recalled Putin's words that he could leave the post of president at the end of his term.
READ MORE: Russian Election Commission Head Hands Over President-Elect Certificate to Putin
The presidential election took place in Russia on March 18, with Vladimir Putin securing a landslide victory, winning 76.69 percent of the votes with the total turnout of 67.54 percent.


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Despite their potential for telemedicine in diabetic foot ulcer treatment, diagnostic accuracy of assessment of diabetic foot ulcers using mobile phone images is unknown. Our aim was to determine the validity and reliability of remote diabetic foot ulcer assessment using mobile phone images.
mHealth Evidence is expanding to include grey literature in the growing body of evidence.
Registered users of mHealth Evidence may now use this bookmarklet feature to contribute material for submission. Please note contributors must be logged in to access this feature. All submissions will be reviewed.


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It would be great to get to know fellow car and bike enthusiasts better. Do take a moment to introduce yourself to the rest of the forum before you continue to post, as this builds a lot of credibility when we can put a name to the username . Follow this simple format below:
Real name:
Occupation (optional):
Cars / Bikes I own (optional):
Interests (optional):
No announcement yet.
Member introductions
This is a sticky topic.
new posts
Member introductionsTags: None
- Stuck
I am Milind Diwate, founder of my latest startup Sahaya Auto Services. Prior to venturing into entrepreneurship with my own Auto Repair Services, I have worked extensively in the industry and accumulated 30 years of professional experience. I am delighted to be a part of the Zigwheels community, and possibly help people seeking certain answers related to car maintenance, repair and other aspects of an automobile industry. I look forward to contributing actively here. Thank you!
Last edited by Sahaya_Auto_Services; 08-03-18, 04:34 PM.
- 1 like
Leave a comment:
Hello JijoMalayil, thanks bro, I'm doing the final editing these days so hopefully in a month or two :-)
Thanks @Joymrc it is a labour of love so hope to do justice with the full trailer, and I hope India has the stomach for music focused travel documentaries ;-) peace
- 1 like
Leave a comment:
Psi_Pixel Glad to have you on the Forum. Looking forward to a variety of travelogues, riding experiences and tips. Eagerly awaiting your biker movie, loved the trailer by the way When is it coming out?
- 1 like
Leave a comment:
Hello Folks,
I'm a biker from Mumbai. I work as a Philanthropy Specialist at international charity animal rights charity, PETA but artist and biker in spirit
I currently ride a Royal Enfield Classic Chrome 500 which I modified after 30,000 kms to 535 cc using a GT piston. My tours include Two Delhi- Ladakh ( one solo), Delhi To Varanasi, Delhi to Goa and sveral tours to Rajsathan. Glad to be a part of this community! I'm in to rock climbing, camping and biking as a traveler, staying in hostels, peoples homes and open grounds.
Currently working on my first biker film. Please see trailer below,
Peace and light
Prashant a.k.a Psi Pixel
- 7 likes
Leave a comment:
Username: Ashster
Real Name: Ashwin Ravindran
Location: Chennai
Occupation: Entrepreneur
My Rides: Hyundai Verna
Interests: Gaming, Reading
A little about myself: I'm a mechanical engineer and I'm currently working on my own startup which is working on connected cars and IOT. Would love to share my product to fellow motor heads for free and get their views and feedback.
- 4 likes
Leave a comment:
Hello This is Vikas Gupta, I am from Chandigarh. I love to travel and see places. I own a Honda Amaze and a Mahindra Quanto. Thanks
- 2 likes
Leave a comment:
Username: Morpheus
Real name: Shivaram Arun
Location: Hyderabad
Occupation: PhD Scholar
Cars I own: Hyundai i20
Interests: Driving, Reading, Cycling
- 1 like
Leave a comment:
Username : vinodkalotra
Real Name : Vinod Kalotra
Location : Pune
Occupation : Security Services
Cars I Own : Alto LXI
Interests : Cars, Bikes, Travelling, Driving, Photography, Trying different cuisines.
- 1 like
Leave a comment:
Username : Akshat Pokhriyal
Real Name : Akshat Pokhriyal
Location : Hyderabad
Occupation : Software Engineer
Cars I Own : Skoda Octavia 1.9
Interests : Cars, reading, travelling, eating, coffee.
- 1 like
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Hello All!
Username: rajnish
Real Name: Rajnish Bhatia
Location: Near Amritsar, Punjab.
Occupation : Retired Architect
Cars / Bikes I own: Getz 1.3L Petrol (2005)
Interests: Cars, Cricket, Meditation.
- 1 like
Leave a comment:
Hi All,
Username: Naree
Real name: Naresh
Location: Bangalore
Occupation (optional): Software Engineer
Cars / Bikes I own (optional): Bajaj Pulsar 150cc , Mahindra Classic 4x4
Interests (optional): Travel , Jeeps
- 2 likes
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I missed this post . Better Late than never...
Username: JOY M.R.C
Real name: JOY M R CHANDRAN
Location: Thiruvananthapuram,Kerala
Occupation: Finished my Previous Project,Looking for a job i Love
Cars / Bikes I own: A Super-Rare 2014 Premier Rio CRDi4 Lx,2005 Maruti Esteem VXi,1995 LML-Vespa Select 150 ES
Interests: Cars,Cars and more Cars,Food,Gaming,Driving,Music,DIY stuff's
- 1 like
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Hi All,
Username: Biju Jose
Real name: Biju Jose
Location: KL 07
Occupation : IT Consultancy / Support
Cars / Bikes I own : FIAT Linea MJD / TVS Apache 150
Interests : Long Distance Driving, Photography, Cars, Social Networking.
Fb : https://fb.com/BIJUJ0SE
- 2 likes
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text:
Parliamentary finance: the expenses of the delegations of the three presidencies waste money .. One of the ministers spent 700 million private trips
15:41 - 02/06/2019
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Information / Special ...
The parliamentary Finance Committee revealed on Sunday that a large waste of public money caused by the delegations of the three presidencies during their travels outside the country, while the statement that one of the ministers spent 700 million Iraqi dinars on his private trips without commissioning work and official functions.
"The delegations accompanying Prime Minister [You must be registered and logged in to see this link.] President Barham Salih and Parliament Speaker Mohammed Halbusi during the foreign visits waste money, unnecessary and larger than the tasks of the visit and does not cover the results," said a member of the Committee Hanin Qaddo said in a statement to / "These delegations cost the state billions of dinars and exhausting the state budget, which lives a kind of austerity and lack of funds."
He pointed out that "all the visits by the three presidencies have not been briefed by the House of Representatives and the extent of the agreements and results achieved from them," explaining that "the Finance Committee is currently working to collect information on the costs of delegations to be discussed within the House of Representatives."
He added that "the House of Representatives is in the process of committing the three presidencies not to accompany large delegations during the official visits and limited to a few of them to reduce the expenses," noting that "one of the ministers in the current government spent more than 700 million Iraqi dinars on private trips without commissioning work and official tasks" .
The three presidencies have conducted a lot of official visits during the past month to a number of Arab and European countries to strengthen relations and economic agreements, as well as moves towards the American side and Iran to ease the tension, accompanied by the three presidencies of a large number of government figures at the level of minister and deputy director general and agent .End 25 T
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text:
Ashleigh Murray, who was previously known by her role as Josie in "Riverdale," yes now changed in the same part to the spin-off "Katy Keene." In an interview, she talked about the differences between the shows and where the potential for cooperation lies.
To further her singing career, Josie (Ashleigh Murray) left her friends and her hometown "Riverdale" with a heavy heart. Their story is not told, as we have known for some time. This will continue in the spin-off of the hyped series "Riverdale" called "Katy Keene."
In an interview with ET, the actress now talked about her new show and how different it is from the previous one: "I love the show so much that she's so light and hopeful, that's what Riverdale lost a lot Totally, but it's on its way."
And with that, the actress is not entirely wrong; after all, the Netflix show became more and more dangerous, dramatic and brutal. But there is also the potential to work together.
So far, it has already been rumored that Archie from "Riverdale" and Sabrina from "Sabrina" become a couple and thus it comes to the great crossover of the two shows that play in the same series universe. Therefore, the assumption is justified that it could also co-operate with the spin-off "Katy Keene."
The former "Riverdale" Actress thinks that exactly on these serious topics and the like a co-production of the two shows could begin.
Besides, Ashleigh also hopes that Veronica (Camila Mendes), her friend from Riverdale, will visit her in New York. Finally, this previously lived in the metropolis. Then we'll be curious to see what's coming! A little more about her role and the series, she reveals in the interview that you can see here completely.


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Back to all reports
TV and children's toy left in bush
Reported via Android in the Flytipping category anonymously at 21:51, Thu 26 July 2018
Sent to North East Lincolnshire Council 3 minutes later
A flat screen TV with stand and children's sit on you have been dumped in a bush at the Patrick Street entrance to People's Park.
Provide an update
Please note that updates are not sent to the council. Your information will only be used in accordance with our privacy policy


text:
Millennials are the generation to really focus on at the moment. They are educated, tech-savvy, information hungry, ambitious individuals. In this article, Kevin Connelly speaks about how you can market to millennials.Read More
Online marketing is a crucial part in running a successful business and an even bigger part in growing that business. In this article, Jeremy K Streeter talks about where we go wrong in terms of online marketing and how we can improve to convert the business.Read More


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Reiki - Great Hucklow
Great Hucklow is a small village and civil parish in the Derbyshire Peak District which nestles under Hucklow Edge between the villages of Tideswell and Bradwell. Wikipedia
Heather Hull Reiki Master Teacher & Practitioner
Dronfield, Derbyshire, S18 7WT
0114 2891342
Having learnt Reiki for myself in 1998 I have been practising Reiki professionally since 1999 and am a member of The Reiki Association. I offer [...]Distance: 6.05 miles.
The No.1 Pain Relief Clinic
Buxton, Derbyshire, SK17 7LF
01298 600477
6 Church View
As a Grand Master for over ten years, I have witnessed many of the benefits of healing and relaxing, Reiki can offer. Everybody is different [...]Distance: 7.04 miles.
Spirit Readings and Reiki
Buxton, Derbyshire, SK17 9EX
01298 436269
Sensitive and compassionate sessions for your highest good. Diane offers one hour reiki sessions in a relaxed, personal setting, working in a sensitive and [...]Distance: 7.81 miles.
Prana Energy Therapies
Sheffield, South Yorkshire, S10 5TR
I help people to lead lives free of emotional and physical discomfort. If you don't want to take medication or feel that traditional healing [...]Distance: 9.93 miles.
Reiki Practitioner
Sheffield, South Yorkshire, S11 7GU
I am a Reiki master using the Usui method and I have been attuned for the past 7 years. I heal individuals and animals for [...]Distance: 10.19 miles.
Reiki Practitioner
Glossop, Derbyshire, SK22 2NJ
0808 1185 696
Serenity Self development offers 1:1 Personal and spiritual development sessions to individuals who are interested in their own personal growth. Issues include: relationships, parenting, [...]Distance: 10.41 miles.
Riverside Wellbeing, Whaley Bridge
Whaley Bridge, Derbyshire, SK23 7LP
0758 1320 036
Thank you for visiting my site! I don't know if you have had a Reiki treatment before, or whether in fact, you are [...]Distance: 10.53 miles.
Healing Light
Sheffield, Cornwall, S10 3BS
I started practicing Reiki when I was 17 years old, and ever since then it has been a way of life for me. I [...]Distance: 10.55 miles.
Time out Therapies
Sheffield, Cornwall, S18 1UA
Reiki, reflexology, massage,facialsDistance: 11.15 miles.
Moria's Place
Sheffield, Cornwall, S6 5JG
Moria's Place is home of the resident Reiki Practitioner and Master/Teacher at The Synergy Centre, Sheffield. At Moria's Place you can receive Reiki Healing, support [...]Distance: 11.42 miles.
Angelic reiki Master practitioner
New Mills, Derbyshire, Sk224hd
My name is Charlotte I am an Angelic Reiki Master practitioner based in the New Mills area of High Peak. I am honoured to [...]Distance: 11.68 miles.
Contemplate Yoga & Well-Being
Sheffield, Cornwall, S6 4WB
Reiki is an ancient healing technique used to help re-balance our body\'s subtle energy (sometimes referred to as Chi or Prana). The treatment is [...]Distance: 11.7 miles.
Crystal Clear Therapies
Sheffield, Cornwall, S35 0PD
Crystal and Reiki Healing. Crystal healing involves working with the chakra's and meredians. Reiki master - healing and attunements courses offered in [...]Distance: 12 miles.
Relaxing Reiki
Hillsborough, County Down, S6 2AT
Have a relaxing treatment to relive tension and everyday stress.Distance: 12.05 miles.
In Perfect Harmony
Sheffield, South Yorkshire, S1 2Ef
I am a qualified reiki master. I practice the Usui system of natural healing. I offer reiki treatments and distant healing. I also offer [...]Distance: 12.36 miles.
inperfect harmony
Sheffield, South Yorkshire, S1 2EG
I am a Reiki master/teacher offering reiki treatments and classes in sheffield.Distance: 12.36 miles.
Powerful Reiki Sessions
Buxton, Derbyshire, SK17 0HH
Join Susan Green, Reiki 1, 2 and Karuna Reiki master, for a powerful Reiki healing session at her magical home in the heart of [...]Distance: 12.4 miles.
Therapeutae Spiritual Reiki
Chesterfield, Derbyshire, S41 8RN
My name is Gary Jones, I am Welsh and a natural healer.. I have practised healing in the UK for over fifteen years becoming [...]Distance: 12.88 miles.
Nurture Me-Complementary Therapies With Nicola Marsh
Chesterfield, Derbyshire, S40 3HL
Reiki Master TeacherDistance: 13.28 miles.
Reiki 2nd Degree Course Traditional Usui Reiki
Oct 2019
Holistic Healthcare, Dronfield Derbyshire
Learn how to use the second level of this powerful form of energy healing to enhance the health and well-being of yourself, friends and family! REIKI 2 PRACTITIONER COURSE IS [...]
Seichem Level 2 Practitioner Training
Nov 2019
Private Location, Rotherham South Yorkshire
The Second Degree Seichem builds upon your previous learning and further enhances the energies enabling you to work as a Seichem Practitioner. Prerequisite: Seichem Level 1 During the course of [...]
Reiki 1st Degree Course Traditional Usui Reiki
Nov 2019
Holistic Healthcare, Dronfield Derbyshire
Learn how to use this powerful form of energy healing to enhance the health and well-being of yourself, friends and family! Reiki 1 is usually learnt as part of a [...]
Reiki 2nd Degree Course Traditional Usui Reiki
Nov 2019
Holistic Healthcare, Dronfield Derbyshire
Learn how to use the second level of this powerful form of energy healing to enhance the health and well-being of yourself, friends and family! REIKI 2 PRACTITIONER COURSE IS [...]