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It is known that physical linkage of TLR ligands and vaccine antigens significantly enhances the immunopotency of the linked antigens. We have used this approach to generate novel influenza vaccines that fuse the globular head domain of the protective hemagglutinin (HA) antigen with the potent TLR5 ligand, flagellin. These fusion proteins are efficiently expressed in standard E. coli fermentation systems and the HA moiety can be faithfully refolded to take on the native conformation of the globular head. In mouse models of influenza infection, the vaccines elicit robust antibody responses that mitigate disease and protect mice from lethal challenge. These immunologically potent vaccines can be efficiently manufactured to support pandemic response, pre-pandemic and seasonal vaccines.
Citation: Song L, Nakaar V, Kavita U, Price A, Huleatt J, Tang J, et al. (2008) Efficacious Recombinant Influenza Vaccines Produced by High Yield Bacterial Expression: A Solution to Global Pandemic and Seasonal Needs. PLoS ONE 3(5): e2257. https://doi.org/10.1371/journal.pone.0002257
Editor: Douglas F. Nixon, University of California San Francisco, United States of America
Received: April 2, 2008; Accepted: April 7, 2008; Published: May 21, 2008
Copyright: © 2008 Song et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work is supported by the VaxInnate Corporation.
Competing interests: All the authors are employees of the VaxInnate Corporation.
Influenza is one of the major infectious disease threats to the human population. It affects individuals of all ages, causes repeated infections throughout life, and is responsible for recurrent seasonal epidemics as well as periodic global pandemics of varying severity. Vaccines are central both to the effective control of seasonal outbreaks and to pandemic preparedness. Hemagglutinin (HA) has been the key protective antigen in seasonal influenza vaccines for the last forty years. While its structure and the basis of its efficacy are well understood, the genetic variability of HA coupled with current methods of vaccine production make it exceedingly difficult to simultaneously meet seasonal and pandemic needs on a global basis. HA changes antigenically to evade the immune response and on average, the prevalent influenza strains in circulation will acquire three to four amino acid changes per year in HA, mostly in regions of HA that are recognized by protective antibodies. Mutations accumulate over time and approximately every three to five years the virus evolves into an antigenically distinct strain [1]. This requires regular updates of the vaccine strains. Additionally, influenza vaccines are typically produced in eggs via a process that takes place nearly year round. Therefore, worldwide production capacity for influenza vaccines is continuously dedicated to the production of seasonal vaccines while pandemic preparedness, either in response to an emerging pandemic or for the generation of a stockpile, requires the redirection of manufacturing resources to the production of a pandemic vaccine at the expense of the seasonal vaccine.
The current inter-related nature of seasonal and pandemic vaccine production has led to intense interest in the development of innovative technologies which could support both seasonal and pandemic influenza vaccine production. Improvements in influenza vaccine production by the industry have recently focused on cell culture. This approach alleviates the significant manufacturing issues associated with egg based manufacturing, but does not improve production efficiency. The intense focus on cell culture production stems from the historical view that protective forms of HA antigens must be manufactured using eukaryotic cells, like those of humans and chickens. The reason for this is that HA undergoes host cell dependent post-translational modification and even though the location and number of different glycosylation sites are not conserved among HAs, it is thought that glycosylation aids in correct folding of the molecule [2]. More recent data, however, show that the glycosylation pattern of HA does not impact the antibody response, suggesting that glycosylation is not required for appropriate folding of the molecule [3].
In addition to improvements in vaccine production efficiency, enhancement of the immunopotency of influenza vaccines will be required in order to meet seasonal and pandemic needs on a global scale. It is now well established that physical linkage of Toll-like receptor (TLR) ligands and vaccine antigens enhances the immunopotency of the linked antigen. TLRs are expressed on various cell types, including professional antigen presenting cells (APC), where they act as primary sensors of microbial infection and then activate signaling pathways that lead to the induction of immune and inflammatory genes. TLR agonists are molecules such as lipoproteins, lipids, sugars or nucleic acids that are specifically associated with pathogenic organisms. Engagement of TLRs by their cognate agonists and the subsequent signaling within APC leads to enhanced processing and presentation of antigens that are co-delivered to those APC [4], [5]. Recently, we demonstrated that the physical linkage of vaccine antigens to the Toll-like receptor 5 (TLR5) ligand, flagellin, generates a significantly more potent vaccine than simple mixing of antigen and flagellin[6], [7], [8].
We here present an approach that addresses many of the production and immunopotency barriers currently associated with seasonal and pandemic influenza vaccines. We have identified a single domain based on the globular head domain of HA which is a self-sufficient protective subunit that can be produced using prokaryotic expression systems. This globular head domain spans the majority of the neutralizing epitopes in HA and stably refolds to faithfully form these conformationally sensitive epitopes. We have genetically fused the globular head subunit to the TLR5 ligand flagellin to create an immunologically potent, highly protective vaccine that is very efficiently manufactured. The increased production efficiency associated with these vaccines means that they can be produced to meet national and even global needs in a period of several months with minimal investments in manufacturing infrastructure.
Rational Design of Globular Head Constructs
Structural studies have shown that two polypeptides, HA1 and HA2, form the monomeric subunit of the HA trimer. The HA1 polypeptide extends up from a membrane proximal stalk, spans the globular head domain and then returns to the stalk. Based on the architecture of HA1, we designed a subunit vaccine which encompassed the neutralizing epitopes of the globular head and also contained the structural elements necessary for spontaneous and efficient folding to correctly display these epitopes after recombinant protein expression in E.coli. The X-ray crystal structure of A/Puerto Rico/8/34 (PR8) was used to guide the design of three prototypic constructs [9] (Figure 1 A–C). In the PR8 crystal structure the HA1 polypeptide has a long tail that interacts with the central coiled-coil formed by the HA2 subunit. The tail is loose and extended, a structural feature that could complicate expression and refolding if included in a subunit construct. By comparison, the globular head domain of HA1 is compactly folded. The head is formed by multiple secondary structural elements that include a β-pleated sheet on the top of the head with tightly packed short α-helices underneath. A second, three-stranded β-sheet lies underneath the α-helices on the membrane-proximal side. An additional small β-sandwich is situated underneath this second β-sheet. Non-structured peptides connect the second β-sheet and the small β-sandwich. These linking peptides do not function as stabilizing elements to the tertiary structure of the globular head, and thus provide ideal sites to truncate the globular head from the rest of HA1 such that it folds independently. We hypothesized that interactions between the α-helices and the second β-sheet, aided by a disulfide bond between the third and fourth conserved cysteines of HA1, are the minimal structural elements necessary for the stable, independent folding of the head domain and maintenance of native, neutralizing epitopes. Therefore, in the design of the first prototypic construct, HA1-2, the boundary was placed in these linking peptides at residues K62 on the N-terminus and S284 on the C-terminus (Figure 1C). Two conserved disulfide bonds are preserved in this molecule.
A) Ribbon diagram of the trimeric PR8 HA0 ectodomain with a monomeric subunit of the HA trimer circled. B) Ribbon diagram of a monomer with the globular head circled. C) Ribbon diagram of the globular head with the boundaries of the HA1-1, HA1-2 and HA1-3 constructs indicated by crosses. Each construct is presented in detail to the right. The beginning and ending residue numbers, in PR8, for the three constructs are labeled. The important secondary structure elements, such as β-sandwich in HA1-1 and the closing β-sheet in HA1-2 are also marked.
We also recognized that inclusion of the small β-sandwich could be required to further stabilize the globular head domain subunit. Therefore, the second prototypic construct, designated as HA1-1, includes this additional secondary structure, and is similar to the thermolysin released fragment previously described by Bizebard et al [10]. The domain boundary for HA1-1 (PR8) was placed between residues S53 and R324 (Figure 1C). The resulting construct contains four conserved disulfide bonds.
In order to evaluate the importance of secondary structural elements such as the second β-sheet and the small β-sandwich in the stabilization of the independent head domain and the consequent display of conformational epitopes, we designed a third PR8 construct, HA1-3, as a control. The boundary for HA1-3 (PR8) was placed at residues N101 to G276 to form a construct which is similar in design and size to an HA subunit previously reported by Jeon and Arnon [11]. The HA1-3 boundary eliminates the β-sheet underneath HA1-2 and all but one conserved disulfide bond.
Each of the globular head constructs were recombinantly linked to the C terminus of Salmonella typhimurium type 2 flagellin (STF2) and the resulting fusion proteins were designated as STF2.HA1-1 (PR8), STF2.HA1-2 (PR8) and STF2.HA1-3 (PR8) according to their decreasing length.
Expression and Conformational Integrity of Flagellin Fusion Proteins
The STF2.HA1-1 (PR8), STF2.HA1-2 (PR8) and STF2.HA1-3 (PR8) fusion proteins were expressed using standard E. coli cell culture. All three proteins expressed equally well following induction. Purified protein was denatured, refolded by rapid dilution and analyzed by SDS-PAGE and western blot under reducing and non-reducing conditions. The Coomassie stained gels show that the purified STF2.HA1-2 (PR8) and STF2.HA1-3 (PR8) proteins were homogeneous. STF2.HA1-1 (PR8) had a predominant band migrating at the correct size and a minor band migrating at a higher apparent molecular weight (Figure 2A).
STF2.HA1-1, STF2.HA1-2 and STF2.HA1-3 proteins were expressed and purified. Refolded proteins were analyzed by SDS-PAGE and Western blot analyses. A) Coomassie stained gel showing the proteins run in the presence (R) or absence (N) of reductant. Bands of the appropriate molecular weight were observed for each construct. B) Western blot analyses using the anti-flagellin monoclonal antibody, 6H11. C) Western blot using PR8-specific immune sera raised following a sub-clinical infection of mice with the PR8 virus.
To evaluate the conformational integrity of the HA subunit of the fusion proteins, Western blots of the SDS-PAGE gels were probed with either the flagellin-specific monoclonal antibody, 6H11, which recognizes a linear epitope (Figure 2B) or convalescent mouse sera raised following a low dose challenge of mice with PR8 virus (Figure 2C). Reactivity of the convalescent sera with protein run in the absence versus the presence of reductant was used as a measure of conformational integrity of the HA moiety. The results show that while 6H11 reacted equally well with all proteins before and after the addition of reductant, the PR8 convalescent serum reacted strongly with the non-reduced forms of STF2.HA1-2 (PR8) but very weakly with the reduced forms, indicating that the majority of epitopes recognized by HA-specific antibodies in the convalescent anti-sera are conformational, and reliant on disulfide bonding. The residual reactivity with the convalescent sera following the addition of reductant is likely due to the small portion of polyclonal antibodies that recognize linear epitopes rather than conformational epitopes. These results demonstrate that the globular head domain correctly refolds during the refolding and purification of STF2.HA1-2 (PR8) protein. A similar pattern of reactivity was observed for the major band of STF2.HA1-1 (PR8). Interestingly, the minor, slower-migrating STF2.HA1-1 band co-migrates with the reduced form of the protein and reacts poorly with the convalescent sera, suggesting that this band corresponds to misfolded protein. In contrast to STF2.HA1-1 and STF2.HA1-2, the convalescent sera reacted poorly with both the non-reduced and reduced forms of STF2.HA1-3 (PR8), indicating that the globular head domain in this recombinant protein is misfolded under the conditions tested. This is consistent with our hypothesis that secondary structures positioned near the peptides linking the globular head domain to the stalk are likely required to ensure stable refolding of the molecule.
To further evaluate the conformational integrity of the HA moiety of the STF2.HA1-1 (PR8), STF2.HA1-2 (PR8) and STF2.HA1-3 (PR8) fusion proteins, ELISA plates were coated with serial dilutions of the full length HA (ecto-domain, HA0, produced in Hi5 cells), PR8 virus and the fusion proteins. The coated plates were probed with either naïve or convalescent mouse sera (Figure 3). As expected, none of the proteins reacted with naïve sera (data not shown). PR8 convalescent sera reacted with PR8 virus and ecto-domain HA0 slightly better than STF2.HA1-1 and STF2.HA1-2 due to their additional epitopes. STF2.HA1-1 was equally antigenic as STF2.HA1-2 despite the minor contamination of the slower migrating band. Consistent with the western blot results shown in Figure 2C, STF2.HA1-3 demonstrated the least reactivity with the convalescent sera.
ELISA plates were coated with the 0.2 µg/well of indicated STF2 fusion proteins, PR8 virus or the full length PR8 HA0 ectodomain expressed in Hi5 cells. Plates were probed with either naïve or PR8 convalescent sera at indicated dilution. Following incubation with HRP-conjugated goat anti-mouse IgG, plates were developed with UltraTMB substrate. Results reflect the delta value of OD450 (Convalescence-Naïve) of samples performed in duplicate. Naïve values (data not shown) were below 0.02.
An in vitro assay based on a cell line that expresses TLR5 and secretes TNF-α in response to TLR signaling was used to assess the functional integrity of the flagellin moiety. In this assay, all three fusion proteins induced strong TNFα secretion, indicative of potent TLR5 activity (EC50≤10 ng/ml for each protein). The endotoxin level for each protein was less than 0.05 EU/µg as measured by LAL assay. These results demonstrate that the flagellin moiety remains functional in the context of the fusion protein.
Taken together, the results confirm the importance of secondary structure in the appropriate refolding of the HA globular head domain. While both the STF2.HA1-1 and STF2.HA1-2 protein fold properly, the STF2.HA1-2 recombinant protein folds more efficiently under the conditions tested. The globular head component of STF2.HA1-3 fails to fold efficiently.
Conformational Integrity of Neutralizing Epitopes
The conformational integrity of defined antigenic regions of the PR8 globular head was tested using a panel of monoclonal antibodies known to be specific for neutralizing epitopes located in the globular head [12], [13]. ELISA plates were coated with either PR8 virus or the fusion proteins STF2.HA1-1 (PR8), STF2.HA1-2 (PR8) or STF2.HA1-3 (PR8). Plates were probed with neutralizing monoclonal antibodies representing each of four previously defined antigenic regions of PR8 (Sa, Sb, Ca, Cb, Figure 4A). As shown in Figure 4B-4E the STF2.HA1-1 (PR8), STF2.HA1-2 (PR8) proteins and the influenza virus reacted comparably with the panel of monoclonal antibodies. There is some reduction in reactivity for the STF2.HA1-1 (PR8) protein which is likely due to the presence of misfolded molecules in the preparation, in agreement with the western blot and convalescent serum ELISA data presented above. The STF2.HA1-3 (PR8) protein failed to bind the monoclonal antibodies.
A) Ribbon diagram depicting of the known antigenic regions of the HA globular head. B–E) ELISA plates were coated with either PR8 virus or the STF2 fusion proteins and probed with a mAb specific for the Sb, Sa, Ca1, Ca2 and Cb region. All STF2 fusion proteins are produced from E coli. F–I) ELISA plates were coated with STF2.HA1-2 and competed against soluble form of HA1-1, HA1-2, STF2.HA1-2, STF2.HA1-3 in binding of the panel of monoclonal antibodies (H37-64, 18 ng/ml; H37-77, 8 ng/ml; H36-11, 188 ng/ml and H163, 500 ng/ml). Bound antibodies were detected by 450 nm absorption. HA1-1 and STF2.HA1-1 were produced in insect cell culture. Both proteins have C-terminal 6His tag. HA1-2, STF2.HA1-2 and STF2.HA1-3 were produced in E. coli. HA1-2 has 6His tag at C-terminus.
The structural integrity of these neutralizing epitopes was further examined using competition assays. Since the panel of monoclonal antibodies reacted equally well with plate-bound STF2.HA1-2 (PR8) and virus particles, the ELISA plates were coated with the STF2.HA1-2 (PR8) fusion protein. The panel of monoclonal antibodies was incubated with the HA1-2 globular head protein alone, STF2.HA1-2 or STF2.HA1-3 for 2 hours. HA1-1 globular head alone or the STF2.HA1-1 protein produced in baculovirus (HA1-1 bv) were included in the evaluation. The mixture was transferred to the ELISA plates and the specific monoclonal antibody reactivity was measured following washing and blocking of the plates. The results (Figure 4F–I) show that HA1-1bv, STF2.HA1-1bv, HA1-2, and STF2.HA1-2, but not STF2.HA1-3 proteins compete for binding of the monoclonal antibodies to plate-bound STF2.HA1-2. These results demonstrate that HA1-1 and HA1-2, either in the context of the fusion or expressed alone, fold properly and that the neutralizing epitopes are correctly displayed. The results further confirm that the STF2.HA1-3 protein fails to stably refold.
Immunogenicity and Efficacy of STF2.HA1-2 in Mice
Groups of 10 BALB/c mice were immunized on days 0 and 14 with 3, 0.3 and 0.03 µg of STF2.HA1-2. A group of naïve mice was included as a negative control. On day 10, animals were bled and the sera of individual animals examined for HA-specific IgG by probing ELISA plates coated with the PR8 virus (Figure 5A). Pooled convalescent sera were included as a positive control. The results demonstrate that immunization with as little as 0.03 µg of STF2.HA1-2 induced measurable levels of HA-specific antibodies post the priming immunization. Mice were subsequently challenged on day 28 with 1×LD90 of the mouse-adapted influenza PR8 virus delivered intranasally. Survival and weight loss were followed for 21 days (Figure 5B and C). As shown in Figure 5B, naïve mice exhibited signs of infection as early as 4 days post-challenge and 90% of the animals succumbed to the lethal challenge by day 21. In contrast, 100% of mice immunized with 3 µg of STF2.HA1-2 (PR8) and 90% of mice immunized with 0.3 µg of STF2.HA1-2 (PR8) survived the challenge. Mice immunized with 3 µg of STF2.HA1-2 (PR8) exhibited no signs of disease as measured by weight loss while mice immunized with as little as 0.3 µg exhibited only very mild weight loss (Figure 5C). Forty percent of mice immunized with 0.03 µg were protected against the lethal challenge. These results demonstrate that E. coli expressed STF2.HA1-2 induces an HA-specific immune response that successfully protects BALB/c mice from a lethal challenge with influenza A virus.
BALB/c mice (10/group) were immunized on day 0 and 14 with 3, 0.3 or 0.03 µg of STF2.HA1-2. A group receiving formulation buffer alone was included as a negative control. A) Sera were harvested on day 21 and evaluated for HA-specific antibody responses by ELISA. B–C) On day 28, mice were challenged i.n. with 1×LD90 of influenza PR8. Survival (B) and weight loss (C) of individual mice were monitored for 21 days post-challenge.
Application to a Currently Circulating Influenza A Strain
The same principles of design were applied to the HA of the currently circulating seasonal strain A/Solomon Islands/3/2006 (SI). Protein was expressed and purified using the methodologies as for the prototypic PR8 construct. To evaluate the immunogenicity and efficacy of this construct, groups of 10 BALB/c mice were immunized on day 0 and day 14 with 3 or 0.3 µg of STF2.HA1-2 (SI), and bled on day 21. Sera were analyzed for hemagglutination inhibition of SI virus using chicken red blood cells as the target. Geometric mean titers (GMT) were 1∶320 (range 1∶160–1∶640) at the 3 µg dose level and 1∶226 (range 1∶80–1∶640) at the 0.3 µg dose level. Ferret immune sera, raised on natural infection and obtained from CDC, exhibited an HAI titer of 1∶320. To further characterize the potency of the vaccine, 15 BALB/c mice were immunized twice with 10, 1 or 0.1 µg of STF2.HA1-2 (SI). Sera were harvested 1 week post the boosting immunization and evaluated for microneutralization titers. In instances where a mouse adapted strain of the virus is not available, as is the case for this seasonal strain, neutralization titers of ≥1∶40 are generally accepted as a correlate of efficacy. The results are reported as GMT in Table 1. The results show that doses of 1 µg elicit GMT titers well above 1∶40 in the mouse model.
To further evaluate the immunopotency of STF2.HA1-2 (SI), groups of 6 New Zealand White rabbits were immunized twice, i.m., with 15 or 5 µg of STF2.HA1-2 (SI). Sera were harvested 3 weeks post the boosting immunization and tested for microneutralization titers. The results in Table 1 demonstrate that STF2.HA1-2 (SI) elicits robust virus neutralizing titers in the rabbit model. Ferret immune sera, raised on natural infection and obtained from the CDC, was included as a positive control in these assays and found to have a neutralizing titer of 1∶5,120.
We have identified the structural elements necessary for efficient refolding of a protective HA subunit after recombinant protein expression in E.coli. Structural information is available for the H1 [9], H3 [14], H7 [15], H5 and H9 subtypes [16] of influenza A as well as for an influenza C HEF (hemagglutinin, esterase, and fusion glycoprotein) [17]. Comparisons of these structures show that the different HAs, while antigenically distinct, are structurally similar and share a common sub-domain organization. Each HA monomer folds to form a membrane proximal stalk and a membrane distal globular head domain. The globular head stands independently from the central stalk and contains the majority of the neutralizing antibody epitopes [12], [13]. We designed three PR8 HA prototypic constructs to test the hypothesis that secondary structures such as the β-pleated sheets near these linker peptides are sufficient to support the head domain as a self-stabilizing unit that can be engineered and expressed apart from the rest of the molecule.
We find that of the three prototypic constructs evaluated, STF2.HA1-2 provides the level of expression and ease of conformationally correct refolding required to support a truly efficient, scalable manufacturing process. This molecule expressed well in our prokaryotic system and refolded easily using rapid dilution. STF2.HA1-1 also refolded albeit with somewhat lower efficiency than STF2.HA1-2, presumably as a consequence of the additional domain and two additional disulfide bonds. Western blots of STF2.HA1-1 run under reducing and non-reducing conditions and probed with PR8 convalescent sera reveal a band in the non-reduced sample that co-migrates with the main band in the reduced sample suggesting that a significant proportion of the protein remained misfolded. STF2.HA1-3 refolded the least efficiently, most likely due to the absence of secondary structures required for stable refolding. Reactivity with a panel of defined neutralizing monoclonal antibodies further supports this conclusion.
STF2.HA1-2 (PR8) was found to be highly immunogenic and efficacious against a lethal challenge in the mouse model. Mice receiving doses of STF2.HA1-2 (PR8) as low as 0.3 µg were protected against a lethal challenge of virus. These data demonstrate that a subunit of HA based on the globular head domain can be fully protective in a standard mouse lethal challenge model. When the same principles of design were applied to the currently circulating seasonal strain, A/Solomon Islands/3/2006, we found that STF2.HA1-2 (SI) was highly immunogenic in both mice and rabbits. In mice doses of 1 µg elicited geometric mean neutralizing titers of 1∶297 and in rabbits doses of 5 µg elicited titers of 1∶453. Thus, the principles of design for these protective subunit vaccines can be applied to different HA molecules.
A key benefit with this approach is that the STF2.HA1-2 recombinant fusion protein can be made quickly, inexpensively and in quantities sufficient to meet global needs. The efficiency of this technology translates approximately into a 1,000 fold gain in production. As a point of reference, the average yield for cell culture is 3 mg/L; for egg based production, 7 mg/L; for baculovirus recombinant synthetic protein, 13 mg/L and for the standard prokaryotic system described here, 3,700 mg/L. This increase in production capacity, along with the fact that it is carried out in a prokaryotic system, provides an opportunity to address several shortcomings of the current egg-based system. One advantage deriving from increased capacity is the ability to increase the dose of antigen. Studies have shown that persons greater than 65 years of age respond less well to the standard vaccine, and that increasing the dose of HA four to five-fold substantially improves the immune response in this segment of the population. Formulation of a "high-dose" vaccine for the elderly becomes a practical possibility with an unconstrained supply of antigen. A second set of advantages comes from eliminating the growth of virus from the manufacturing process. Currently, vaccine production strains are created by crossing the HA and NA genes from candidate circulating strains onto an egg-adapted virus, generally the PR/8/34 strain. Manufacturers then further adapt these production strains to create high-yield viruses. The adaptation process results in selection of mutations in the upper part of the HA globular head near the receptor binding site [18]. As a consequence, the HA in the vaccine may be different from the HA in the original viral isolate. Prokaryotic expression, based on a cDNA copy of the original isolate HA, avoids genetic selection during the production process. In addition, there are situations where a suitable production strain cannot be created by conventional methods. In these cases, a related HA production strain is pressed into service. Eliminating the need to grow virus allows the production of the desired antigen with the original sequence.
In conclusion, the manufacturing approach described herein has major advantages over existing technologies in that it allows faster molecular development, rapid manufacturing and very high levels of productivity at small manufacturing scales. These advantages are critical to the successful production of seasonal and pandemic influenza vaccines.
Materials and Methods
Cloning of recombinant HA genes
E. coli.
The codon optimized synthetic genes of the hemagglutinin (HA) globular head domain of PR8 were fused to the C-terminus of the full-length sequence of Salmonella typhimurium fljB (flagellin phase 2), STF2 (DNA2.0 Inc., Menlo Park, CA). The sequence SGSGSGS was incorporated at the junction of STF2 and HA as a flexible linker. The resulting fragments corresponding to the STF2.HA1-1, 1-2 and 1-3 genes respectively were cloned to pET24a vector to generate the constructs STF2.HA1-1, STF2.HA1-2 and STF2.HA1-3. The proteins were expressed in BLR3 (DE3) cells (Novagen, San Diego, CA; Cat #69053).
The synthetic genes encoding HA1-1 (PR8) or in fusion with STF2 were codon-optimized for Baculovirus expression (DNA2.0 Inc., Menlo Park, CA) and cloned into pFastBac™. The honey bee mellitin sequence (MKFLVNVALVFMVVYISYIYAD PS) was fused to the amino terminus of recombinant proteins to provide a secretion signal and hexahistidine was tagged to the carboxyl terminus to facilitate purification. The synthetic genes were cloned to the pFastbac1 vector. The recombinant Baculovirus generation followed standard Bac-to-Bac® Baculovirus Expression protocol (Invitrogen, Carlsbard, CA).
Protein production
High expresser clones were cultured overnight and used to inoculate fresh LB medium supplemented with 25 µg/ml kanamycin, 12.5 µg/ml tetracycline and 0.5% glucose. At an OD600 = 0.6 protein expression was induced with 1 mM IPTG for 3 h at 37°C. Cells were harvested by centrifugation (8,000g for 7 minutes) and disrupted by microfluidizer (18,000 psi). The inclusion body was washed with 1% Triton X100 and dissolved in 8 M urea. The filtered protein solution in 25 mM NaCl and 50 mM Acetate, pH 4.0 was applied to a SP Sepharose Fast Flow column (GE/Amersham). The fraction peak was eluted by salt gradient and buffer exchanged to 50 mM Tris, 25 mM NaCl and 8 M urea, pH 8.0. Protein refolding was achieved by rapid dilution (1∶10) into 100 mM Tris-HCl buffer (pH 8.0), and further purified by anion exchange (Source Q, GE/Amersham). For final polishing and endotoxin removal, a Superdex 200 gel filtration column (10/300 GL, GE/Amersham) was used. The protein peak was eluted using 100 mM Tris, 150 mM NaCl, 1% glycerol and 1% Na-deoxycholate elution buffer. Peak fractions were pooled, dialyzed against 1×PBS and stored at −80°C. For all 6xHis tagged proteins, the metal chelating column was employed. Protein was loaded to a Ni-NTA column equilibrated in 20 mM Tris, pH 8, 0.5 M NaCl and eluted in a gradient of 0–0.5 M imidazole. The target protein was further purified by size exclusion column (10/300 GL, GE/Amersham). The peak fractions were pooled, concentrated and dialyzed against 1×PBS. Aliqoted protein solution was stored at −80°C. Endotoxin contamination was assayed by using standard Chromogenic Limulus Amebocyte Lysate assay (Cambrex, Walkersville, MD) as directed by the manufacturer.
Many aspects of the ELISA methods were held in common. ELISA plates were coated with the indicated proteins in PBS overnight at 4°C or one hour at room temperature. All washes between reagent addition steps were performed 3 times with 1X PBS/0.05% Tween-20. Plates were blocked with 200–300 µl/well of Assay Diluent Buffer (ADB; BD Pharmingen) for 1–3 hour at 23–27°C. After incubation with the indicated detection antibodies, HRP-labeled goat anti-mouse antibody (Jackson Immunochemical) diluted in ADB was added and the plates were incubated at 23–27°C for 1 hour. After adding TMB Ultra substrate (Pierce) and monitoring color development, the reaction was stopped with 1 M H2SO4 and OD450 was measured on a microplate spectrophotometer.
Protein ELISAs.
Plates were coated with serial dilutions of proteins. After block, plates were probed with monoclonal antibody specific for flagellin (6H11; Inotek) or convalescent sera against PR8 virus overnight at 4°C.
Serum antibody determination.
Plates were coated with 100 µl/well HA1-1 produced in insect cells in PBS (5 µg/ml). Dilutions of the sera in ADB were added (100 µl/well) and the plates were incubated overnight at 4°C.
Viral ELISA.
Sucrose density gradient purified PR8 virus (Advanced Biotechnologies Inc.,) or STF2 tagged recombinant HA proteins were diluted to 4 µg/ml in 1X PBS and 100 µl coated in triplicates. After block, plates were incubated with 100 µl of HA-specific antibodies diluted in ADB at 25°C for 2.5 hours.
Competition ELISA.
Plates were coated overnight with 100 µl/well STF2.HA1-2 (PR8) at 2 µg/ml. Antibodies were pre-incubated with serially diluted recombinant proteins for 2 hours at 25°C and added to the washed and blocked STF2.HA1-2 (PR8) coated plates for a further 2 hour incubation followed by detection antibody. The amount of antibody used for each epitope was pre-determined to be in the linear range of a saturation curve in ELISA.
Cells and viruses
MDCK cells were obtained from ATCC and maintained in DMEM supplemented with 5% FBS, 2 mM L-glutamine, 100 units/ml Penicillin and 100 µg/ml Streptomycin. Influenza viruses, mouse adapted A/PR/8/34 and A/Solomon Islands/3/2006, were obtained from Dr. Y. Kawaoka (University of Wisconsin) and CDC, respectively, and propagated in either MDCK cells or 11-day old SPF embryonated hen's eggs (Charles River Laboratories, North Franklin, CT).
TLR5 bioassay
The bioactivity of purified recombinant proteins was tested as previously described [6].
Animal studies
BALB/c mice 6–8 weeks old were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed in either the Yale University vivarium (New Haven, CT) or the Princeton University vivarium (Princeton, NJ). All studies were performed in accordance with the University Institutional Animal Care and Use Committees (IACUC). Recombinant proteins were prepared in one of two vehicles: PBS (phosphate-buffered saline) or formula F147 (10 mM L-histidine, 150 mM NaCl, 5% trehalose, 0.02% polysorbate 80, 0.1 mM EDTA, 0.5% ethanol, 10 mM Tris, pH 7.2). Vehicles were used interchangeably without detectable impact on the results. Mice were immunized subcutaneously (s.c.) on days 0 and 14. On days 13 (primary) and 21 (boost), individual mice were bled by retro-orbital puncture. Sera were harvested by clotting and centrifugation of the heparin-free blood samples.
Studies with female and male New Zealand White rabbits were performed at Covance Research Products (Denver, PA). Rabbits (6/group) were immunized intramuscularly (i.m.) on days 0 and 21 with 5 or 15 µg of STF2.HA1-2. Sera were harvested 3 weeks post the booster and evaluated for HA-specific microneutalization titers.
Microneutralization assays
Serum samples were treated with receptor destroying enzyme II (RDE, Denka Seiken Co., Ltd., Tokyo, Japan) and co-cultivated with 100 TCID50 of influenza virus A/PR/8/34 or A/Solomon Islands/3/2006 for 1.5 hr in series dilution (duplicate). MDCK cells (4×104 /well) in DMEM supplemented with 1% BSA, 20 mM HEPES, and 100 IU/ml Penicillin and 100 µg/ml Streptomycin were then added and incubated for 20 hours at 37°C. Cells were washed, fixed, air-dried and incubated with a monoclonal anti-influenza A nucleoprotein antibody (1∶2,000, clones A1 and A3, ATCC/BEI resources). Signals were detected by OD450. Virus back titration, positive serum control, virus controls (VC), and cell controls (CC) were included in the assay. The end point of virus neutralizing antibody for each serum was determined using 50% of specific signal = [(Average OD of VC wells)–(Average OD of CC wells)]/2+Average OD of CC wells. Values below this value are considered positive for neutralizing activity.
Statistical analyses
The titers of neutralizing antibodies were transformed into natural logarithm, and subjected to ANOVA/Tukey tests. Survival curves between different groups were compared with Log-rank test. Data analysis used GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California USA, www.graphpad.com).
Influenza virus challenge of mice
To assess efficacy, mice immunized on days 0 and 14 as described above were challenged on day 35 by intranasal administration of 1×LD90 (dose lethal to 90% of mice; 1×103 TCID50) of influenza A isolate, PR8. Animals were monitored daily for 21 days following the challenge for survival and weight loss.
We thank Dr. Walter Gerhard (The Wistar Institute) for assisting with the selection of PR8 specific monoclonal antibodies; Dr. Yoshihiro Kowaoka (University of Wisconsin) and Dr. Lynda Cauley (University of Connecticut) for providing PR8 virus. We thank Bruce Weaver, Dallas Jock and Lorine Lantz for technical assistance and Dr. Alan Shaw, Dr. Robert Becker, Dr. Richard Flavell and Dr. Ruslan Medzhitov for helpful discussion and reading this manuscript.
Author Contributions
Conceived and designed the experiments: LS TP LT. Performed the experiments: VN UK AP JH JT AJ GL YH PD VT SU LR RB HL YZ GM. Analyzed the data: LS VN UK AP JH GL SU WW TP LT. Wrote the paper: LS LT.
- 1. Smith DJ, Lapedes AS, de Jong JC, Bestebroer TM, Rimmelzwaan GF, et al. (2004) Mapping the antigenic and genetic evolution of influenza virus. Science 305: 371–376.
- 2. Hebert DN, Zhang JX, Chen W, Foellmer B, Helenius A (1997) The number and location of glycans on influenza hemagglutinin determine folding and association with calnexin and calreticulin. J Cell Biol 139: 613–623.
- 3. Bright RA, Ross TM, Subbarao K, Robinson HL, Katz JM (2003) Impact of glycosylation on the immunogenicity of a DNA-based influenza H5 HA vaccine. Virology 308: 270–278.
- 4. Blander JM, Medzhitov R (2004) Regulation of phagosome maturation by signals from toll-like receptors. Science 304: 1014–1018.
- 5. Blander JM, Medzhitov R (2006) Toll-dependent selection of microbial antigens for presentation by dendritic cells. Nature 440: 808–812.
- 6. Huleatt JW, Jacobs AR, Tang J, Desai P, Kopp EB, et al. (2007) Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid cellular and humoral immunity. Vaccine 25: 763–775.
- 7. Huleatt JW, Nakaar V, Desai P, Huang Y, Hewitt D, et al. (2008) Potent immunogenicity and efficacy of a universal influenza vaccine candidate comprising a recombinant fusion protein linking influenza M2e to the TLR5 ligand flagellin. Vaccine 26: 201–214.
- 8. McDonald WF, Huleatt JW, Foellmer HG, Hewitt D, Tang J, et al. (2007) A West Nile virus recombinant protein vaccine that coactivates innate and adaptive immunity. J Infect Dis 195: 1607–1617.
- 9. Gamblin SJ, Haire LF, Russell RJ, Stevens DJ, Xiao B, et al. (2004) The structure and receptor binding properties of the 1918 influenza hemagglutinin. Science 303: 1838–1842.
- 10. Bizebard T, Gigant B, Rigolet P, Rasmussen B, Diat O, et al. (1995) Structure of influenza virus haemagglutinin complexed with a neutralizing antibody. Nature 376: 92–94.
- 11. Jeon SH, Arnon R (2002) Immunization with influenza virus hemagglutinin globular region containing the receptor-binding pocket. Viral Immunol 15: 165–176.
- 12. Caton AJ, Brownlee GG, Yewdell JW, Gerhard W (1982) The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype). Cell 31: 417–427.
- 13. Wiley DC, Wilson IA, Skehel JJ (1981) Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature 289: 373–378.
- 14. Wilson IA, Skehel JJ, Wiley DC (1981) Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature 289: 366–373.
- 15. Russell RJ, Gamblin SJ, Haire LF, Stevens DJ, Xiao B, et al. (2004) H1 and H7 influenza haemagglutinin structures extend a structural classification of haemagglutinin subtypes. Virology 325: 287–296.
- 16. Ha Y, Stevens DJ, Skehel JJ, Wiley DC (2002) H5 avian and H9 swine influenza virus haemagglutinin structures: possible origin of influenza subtypes. EMBO J 21: 865–875.
- 17. Rosenthal PB, Zhang X, Formanowski F, Fitz W, Wong CH, et al. (1998) Structure of the haemagglutinin-esterase-fusion glycoprotein of influenza C virus. Nature 396: 92–96.
- 18. Kilbourne ED, Easterday BC, McGregor S (1988) Evolution to predominance of swine influenza virus hemagglutinin mutants of predictable phenotype during single infections of the natural host. Proc Natl Acad Sci U S A 85: 8098–8101.


text:
It seems Rep Turner is able to look at a man working and determine whether he is in the country legally. That, my friends, is powerful stuff. I'm sending him to a few hospitals in the area to help ferret out those pesky Canadian nurses.
Seriously, I'm with you that companies performing work and receiving Federal funds should make American citizens a priority. I could even get behind the idea of auditing the I-9s of those companies to ensure the same. But to give credibility to the argument that we can look at someone and determine their immigration status is not only a rookie mistake, it is a dangerous one.

subset: commoncrawl


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Serves 2
Oh fall. Who can resist the flavors of fall? Not me!! So when I am looking for fall flavors with the least possible effort, I turn to smoothies. And, unlike other green smoothies I make, this one is appropriately green because it's green apple!!
I think dates taste like toffee, so the combo of dates and apples reminds me of caramel apples. Yum! You can whip this up for breakfast, or dessert, and get a great helping of leafy greens!!
I didn't share this earlier this week, but this past weekend, I took a day to relax and read. It was amazing. Something about immersing myself in another world, usually with a kick-ass girl as the lead, makes me feel whole and refreshed. It was amazing. But when I get into a good book, I can be really silly and stay up waaay too late reading it. I did that and managed to finish my book in a little over a day. So worth it though, I love reading so much!!
When I needed a little perk up because of my late night reading, this smoothie was the perfect fix. Sweet and filling. Plus I could sip with one hand and read with the other.. hehe
Health highlight of apples: Apples are amazingly healthy. The flavonoids in apples have been shown to reduce the risk of certain kinds of cancer. Apples have also been shown to sharpen your memory, and help protect against Alzheimer's, asthma, and chronic cough. They are protective of your cardiovascular system, and have been linked with a reduced risk of type 2 diabetes. The skin is the most dense with nutrients, so leave it on, but opt for organic as apples are the produce found to have the highest pesticide residue.
¼ cup Dates
2 granny smith Apples (cored)
2 cups Water (almond milk works too)
1 cup Spinach
1 cup Ice
½ tsp Cinnamon
Pinch Nutmeg
- Place all the in the blender
- Blend on high until smooth
- Add more ice if needed
- Drink up and enjoy!!!!!!!
What is your favorite green smoothie?


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Has it affecting anyone's travel plans?
Terroists affecting travel?
Won't be affecting mine, and I'll be in London for a night on the way over to India, and a full day on the way back (that's what happens when you use FF miles to get free tickets, you get terrible connection too). Anyway, I'm excited to spend a day cruising around London and checking out the sights (since I have one full day in London, I'll probably get one of those hop on, hop off, double-decker tour buses, so I'll be an easy target, but no plans to change this plan).
The only place terrorism has affected my travel plans has already been in effect for 4 years now. Otherwise I would have definitely visited Pakistan by now (I'd really love to visit the Northern areas). And probably Yemen as well. I'd still really like to visit both of these places as well as Iran & Afganistan, but looks like they'll all have to wait for a while.
I am allmost going to think that you will be safer in the middle-east then in Europe/Nort-America
Quoting wouterrr
I am allmost going to think that you will be safer in the middle-east then in Europe/Nort-America
That's a good point!
Going to London for a week-end next week. No cowards won´t disturb my plans.
Yeah, Travel100, I think there will be not much difference in safety between the middle-east and Europe.
Look at this example:
You bought your planeticket allready for Karachi (or Teheran...). Then you listened to all the people for not going because of supposed bomb attacks there!!! So, you decided not to go but instead going to London on 6 july and you decide to take the metro on 7 july in the early morning...........
Why should people change their plans? Everyone in London (and everywhere else for that matter) knew it was gonna happen, it was just a matter of when! It is obviously a horrific experience that nobody wants to go through, but if we stop travelling we are yielding to the terrorists plans!
I was living in Switzerland when a madman ran riot in my local governmental building and shot 14 people dead. The whole country went into shock as the country is considered so safe(the year before the whole of Switzerland had recorded a mere 12 murders.). It just goes to show that you can be anywhere in the world and you will always be subject to crime, natural disasters, fatal accidents etc. Its part of life!
PS: Admittedly I will not necessarily be buying a plane ticket to Iraq or Dafur in the near future, but entering a war zone is on a slightly different scale.
Quoting tallbird
PS: Admittedly I will not necessarily be buying a plane ticket to Iraq or Dafur in the near future, but entering a war zone is on a slightly different scale.
Agree very much with you Tallbird, some excellent thinking
Terrosim won't be affecting my travel plans. There are about a thousand things that can happen to you when you're on vacation and terroism should be a really minor consideration. With planning and appropriate cautions, you can keep yourself safe in most destinations.
Quoting CupCake
Has it affecting anyone's travel plans?
Nope, not affecting me. And I doubt it'll affect London too much either. Heck, they lived through the blitz during WWII, they lived through the IRA bombings in the 70s-80s-90s. They'll live through this as well. Rome ain't the eternal city, London is.


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BARCELONA -- An international study confirmed the value of the European Society of Cardiology (ESC) guidelines to determine the risk-benefit ratio for implantable cardioverter defibrillators (ICD) in hypertrophic cardiomyopathy patients.
"We calculated that for every 13 high-risk patients who receive an ICD as recommended by ESC guidelines, one patient could potentially be saved from sudden cardiac death," said Constantinos O'Mahony, MD, of St. Bartholomew's Centre for Inherited Cardiovascular Disease and the University College London Centre for Heart Muscle Disease in London.,
Overall, the HCM-EVIDENCE trial showed that the tool could distinguish well between high- and low-risk patients, with good agreement between what it predicted and their actual 5-year sudden cardiac death rates, O'Mahoney said during a press conference at the ESC meeting.
Specifically, patients classified as low risk were predicted to have a sudden cardiac death incidence of less than 4% at 5 years so had a 5-year sudden cardiac death incidence of 1.4%. Those classified as high risk were predicted to have a sudden incidence of at least 6% at 5 years and had a 5-year sudden cardiac death incidence of 8.9% in the study, O'Mahony reported.
The HCM Risk-SCD has been in place since 2014, he noted. "But, whenever a risk model is created, there are concerns about how generalizable it is outside the original study population. Since we developed the HCM Risk-SCD tool using patients treated exclusively in Europe, it was important for us to demonstrate its applicability in other healthcare systems with different medical expertise and potentially different patterns of disease," he said.
The hypertrophic cardiomyopathy guidelines recommend using the HCM Risk–SCD risk calculator to estimate a patient's 5-year risk of sudden cardiac death, with the recommendation that high-risk patients receive ICDs to prevent fatal ventricular arrhythmias.
For this validation study, O'Mahony's group in North America, Europe, the Middle East, and Asia recruited 3,703 patients from 14 centers. The patients were followed for a median of 5.9 years and during the course of the study, 73 sudden cardiac death events occurred.
"The study also shows that the HCM Risk-SCD calculator can be used to avoid unnecessary ICD implants in low risk patients, supporting the 2014 ESC recommendation not to implant ICDs in these individuals," O'Mahony said.
He noted that despite the good prognostic performance of the HCM Risk-SCD calculator, no strategy will ever be able to predict and prevent all sudden cardiac death cases, especially for intermediate risk patients.
Nancy Sweitzer, MD, director of the Sarver Heart Center at the University of Arizona in Tucson commented that the trial offered real-word evidence of how the ESC guidelines work.
"The study results suggest that in the validation cohort the guidelines performed very well," she told MedPage Today. "The guidelines stratified patients into a low risk group for sudden cardiac death and less than 2%; a moderate risk group of 2-4% and then a high risk group of greater than 6% of sudden cardiac death. In fact, over the course of 3-4 years in the validation cohort, after the score was applied, the high risk cohort does show a substantial risk of sudden death events."
The guideline algorithm identifies a group with substantially elevated risk for sudden cardiac death, which is important for patients that are young, as the complications associated with an ICD at a young age are significant, she noted.
"Accurately identifying that patient at risk is terribly important clinically," she stated. "I think the take away for clinical practice is to become familiar with the [HCM Risk-SCD calculator] for decision making in hypertrophic cardiomyopathy," she said, adding that physicians can download an app to their phone or computer "to help you with a patient at high risk and [determine] whether you should counsel that patient about the risks and benefits of defibrillator implantation."
O'Mahony and Sweitzer disclosed no relevant relationships with industry.
- Reviewed by Robert Jasmer, MD Associate Clinical Professor of Medicine, University of California, San Francisco and Dorothy Caputo, MA, BSN, RN, Nurse Planner
last updated


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Chris' last day here I took him around the area I used to live. I was born in Michigan and have lived the majority of my life here even though I can't believe that this is year 7 living in Utah.
We drove to Birmingham and Bloomfield Hills area where we stopped at Cranbrook Institute which has the top art school of the country. When I was young I worked here for a boys day camp and oh my heck that was a challenge. I had 22- 8-year-old boys. The grounds are so lovely and designed from some of the top designers.
I also went past my high school Seaholm High School and Quarton Elementary and I think its so cool too that my dad and aunt and uncle went there.
We also went to Downtown Birmingham which I love. The building below is the Townsend Hotel where famous people stay. I remember when the Lakers played the Pistons here in Detroit, the Lakers practiced at our school and stayed here while they were in town.
We also had some golf tournaments at Oakland Hills Country Club right behind our old house and when Tiger Woods and a ton of famous people were in town, even the queen and prince, they stayed at the Townsend. We walked inside it's beautiful.
Later on we went to downtown Detroit. This is Compuware where my dad works. This building is so pretty, in Detroit they are trying to build up to make it nicer.
Here is the front, they are a software development company so their slogan is "We make IT rock"... like IT... information technology. I thought it was cool
Here is the inside at an angle. There's a pond at the bottom and water comes down from these things.
This is the DIA... Detroit Institute of Art
This is the old train station which I think is gorgeous. The architecture and detailing is incredible and I didn't know this but this building is in Transformers 1.
Here's us at Mexican Village. So creepy but the food is incredible and as authentic to Mexico as you can get being thousands of miles away
Dang can't remember what this building is but it's my favorite. Again Detroit has some beautiful architecture and was a beautiful city
We were supposed to get another building in the back. Oh well, here is Chris and I in downtown Detroit. 8-mile is the most dangerous along with Cass Ave which is like 1-3 blocks away. Yikes! Chris has my back though
The General Motor building and Renaissance Center. Not sure if those are empty or whats going on but they do look beautiful especially right off the lake at night
Sunday, August 15, 2010
Being Tour Guide of Michigan for Chris
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A favorite pancake recipe of mine. This recipe went through vigorous testing to create an amazingly fluffy and light pancake. The olive oil addition is what creates such a moist cake. Olive oil can be replaced by melted butter, or even browned melted butter, for a richer flavor. Add fresh fruit or citrus zest to your completed batter to create other versions of this beauty.
For more, check out our Basic Pancakes, Blueberry Buttermilk Pancakes, and Sweet Potato Pancakes.


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[Samui hotels]
中 文
日 本
Hathai House
84/61 Moo 3, T. Maret, Lamai Beach
Koh Samui, Suratthani 84310
Hotel Kategorie:
25 Kundenbewertungen :
Zimmer :
Einrichtungen :
Service :
Standort :
Das Hathai House befindet sich im Zentrum von Lamai, in Gehweite zum berühmten Lamai-Strand, und ganz in der Nähe der Unterhaltungsgegend und des Thai-Box-Stadions. Das Hotel spiegelt die Eleganz der thailändischen Kultur wider. Es bietet 10 Standardzimmer, stilvoll eingerichtet mit komfortablen, modernen Möbeln, sanften Farben und geschmackvollen Kunstwerken. Die Zimmer sind ausgestattet mit Dusche, einer Minibar und einem Safe sowie TV und DVD-Player und einer große Auswahl an Filmen steht an der Rezeption zur Verfügung. Das Hathai House serviert köstliche Gerichte im Restaurant neben dem Pool wo Sie authentische thailändische Küche mit frischem Fisch und Meeresfrüchten sowie traditionelle thailändische Speisen genießen können.
Hotel Überblick
Preise & Buchung
Hotel Bilder
Hotels in der Nähe
Travel reviews for Hathai House
Date of stay:
Apr 26, 2017
Accommodation and facilities:
I was very satysfied but I have onething I hope the hotel will get a better tv program so one like my self who don t speak or understand much thai can follow the news and see some films and sport
Reservation service:
Everything went as expected
Apr 28, 2017
written by:
René Jensen
Date of stay:
Aug 16, 2014
Accommodation and facilities:
Great location. Within walking distance to the beach, bars, night market and restaurants but far away not to be disturbed by the music from the bars. Big rooms and great pool. Nice small hotel.
Reservation service:
good service and they went out of their way to accommodate a group of 14 people arriving and leaving on different days.
Aug 19, 2014
written by:
Jennifer Hohls
Date of stay:
Jan 02, 2014
Accommodation and facilities:
Good location, about 5 minutes to the beach, but the beach is not very good. Friendly staff, room service every day. But really overpriced for high season, guess it s real price not more then 25-30$ per night.
Reservation service:
Great service)
Jan 05, 2014
written by:
Date of stay:
Dec 07, 2012
Accommodation and facilities:
great little hotel in lamai centre. rooms are pretty big with a huge decent bed. jimmy the owner is a great host. just five mins walk to the beach around the corner from the night life. recommended great hotel for the price.
Reservation service:
great booking service. recommended
Dec 24, 2012
written by:
mr hiten thakkar
Date of stay:
Aug 29, 2012
Accommodation and facilities:
There are three must haves for me in booking Thai accommodation; pool, air con and room safe. Hathai House ticks all these boxes plus much more. Well located for shops and night life, excellent free wifi, great breakfast and friendly help and service from Noo and her staff. We had a poolside room so cooling off was real easy without the hassle of locking up your room and carting around gear which you need to do in the larger resorts. The only shortcoming (and a minor one) is the absence of tea/coffee making facilities in- room, so just got used to living with my one cuppa in the morning at breakfast!
Reservation service:
Great - no problems at all
Sep 01, 2012
written by:
Brett Sheehan
Date of stay:
Apr 12, 2012
Accommodation and facilities:
Thank you Jimmy with family for ten great days gurra&kjelle
Reservation service:
Apr 20, 2012
written by:
gunnar olsson
Date of stay:
Feb 29, 2012
Accommodation and facilities:
Back for a third time and as before - Jimmy and his entire team made my stay enjoyable. Small boutique basic hotel that offers clean and cosy comfort. Not too far away from the beach - about a 5 minutes walk. Not far from food and entertainment.
Reservation service:
No problems. Prompt. Efficient
Mar 03, 2012
written by:
Date of stay:
Feb 09, 2012
Accommodation and facilities:
Very nice hotel! All u need in a hotel.
Reservation service:
No problem vith booking.
Feb 14, 2012
written by:
Niclas Johansson
Date of stay:
Jan 24, 2012
Accommodation and facilities:
Hathai house is a very nice little hotel close to everything but you are not disturbed by the noises in the street. the owners and the staff are very helpfull and friendly. the room has been nicely cleaned every day. We love our hathai house!
Reservation service:
Thank you very much ms. tiim! the service was very very fast and friendly! i have been a little confuses about the arrival date bur mr. tin checked until everything was fine.
Jan 27, 2012
written by:
Katrin Jacobi
Date of stay:
Dec 04, 2011
Accommodation and facilities:
I was fine. The staff is very welcoming and helpful. Advice to people who want to stay in a simple and quiet.
Reservation service:
Dec 07, 2011
written by:
Emiliano Gusella
Date of stay:
Aug 18, 2011
Accommodation and facilities:
Everything it s ok. I ll come back again SURE..Staff friendly.
Reservation service:
Aug 29, 2011
written by:
Massimo Italy
Date of stay:
Mar 10, 2011
Accommodation and facilities:
Returning guest. Again, Jimmy and his entire team were excellent in making my stay a pleasant one. Clean room. Clean bathroom. Basic amenities provided as always. Nice quiet location away from the hustle and bustle of Lamai beach yet convenient to get to the beach.
Reservation service:
Sawadee was excellent again.
Mar 14, 2011
written by:
THB ฿ Thailand
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Hotels in der Nähe Hathai House
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Lamai is now further enhanced by the newly opened
Hathai House
situated in central lamai within walking distance to lamai beach. The resort reflects a great blend of bali style and the elegance of thai culture.
You can enjoy the early morning experience of stepping straight into our outdoor swimming pool including waterfall or sitting poolside and get a taste of the relaxing life in the tropics. Our restaurant serves Thai and European cuisine.
We offered 10 standard and comfortable, stylishly decorated with contemporary Thai furnishing, tasteful artwork and soft color tones.
:: Standard Room ::
:: Standard Room, Twin Bed ::
:: Pool View Room ::
:: Superior Room, Double ::
:: Family Room ::
:: Deluxe 2 Bedrooms ::
Room Features
Thai furnished
Air conditioning
Cable TV
DVD player (More than 100 films available at the reception for free of charge)
Electronic safety box
Tea & coffee making facilities
Hot shower
:: Restaurant & Bar ::
Hathai House serves delicious meals in the swimming pool restaurant where you can enjoy authentic Thai cuisine including fresh seafood and Thai traditional food. Our restaurant serves Thai and European cuisine. Our staffs will be please to give you all information to make you a pleasant holiday.
Swimming pool waterfall
Wi-Fi Internet
Tour excursion, ticket and booking
Laundry service
Jeep and motorbike rental
Airport transfer
Hathai House
is located in the central of lamai and just 400 meters away from Lamai beach. You will find shops and entertainment area just across the road.
zum Seitenanfang
[Samui hotels]
[Hathai House ]
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Program l
A young writer goes missing after arriving in Brooklyn. Bingham Bryant plays the young man whose friends unsuccessfully try to find him by combing through his notebooks, letters, postcards and other possessions for clues. Running in parallel is a story of a philosopher, filled with mysteries and political statements. This dark, minimalistic story with creative screen composition is peppered with humor, and a limited number of images are repeatedly presented. Simple shots designed to convey information such as postcards, newspaper clippings, and notes are connected and repeated, generating a unique rhythm. Ricky D´Ambrose, known for his short films, makes a bold debut showcasing his exceptional talent. The film takes a dystopian view of the modern world using documents from everyday life and delicately forged texts. [JANG Byungwon]
Born in New Jersey, in 1987. In 2012, he received a Master's degree in film studies from Columbia University, in New York. Since 2013, he has shot and edited a series of video-recorded directors' talks?featuring Chantal Akerman, Bruno Dumont, and Matias Pineiro, among others?for MUBI Notebook. He has written and directed four short films, including Six Cents in the Pocket(2015), which was included in the 53rd New York FF, the 66th Berlinale, and the 2016 Viennale. Notes on an Appearance, completed in 2017, is his first feature.


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BY SAM SPOKONY | Residents are invited to attend a quality of life forum at the Hudson Guild on Monday, April 21 at 7pm, where community leaders and elected officials will discuss neighborhood issues and take questions from audience members.
The forum will be hosted by both the Neighborhood Advisory Committee and the Senior Advisory Council of the Hudson Guild, a social services organization that has been helping Chelsea residents with locally based issues for more than 100 years. Seniors are especially welcomed to come and share their concerns about Chelsea's transforming landscape and their thoughts on what the city administration should focus on for the future.
Topics of discussion will include health, housing, safety, traffic, biking, transportation, noise and weather conditions. A panel of officials including State Senator Brad Hoylman, Manhattan Borough President Gale Brewer, Assemblymember Richard Gottfried and City Councilmember Corey Johnson will open the forum with introductory comments on quality of life, after which they will stay to hear directly from residents.
"New York City changes every day. Those changes can affect your quality of life and it is important to know what services are available to you," said Councilmember Johnson. "The Neighborhood Advisory Committee and Hudson Guild are excellent community resources and I thank them for helping to make sure New Yorkers can make well informed decisions about their lives."


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My mum asked me to clean my drawers and arrange my things. Because they were dirty.
It was hard work and and it took lot of time. But I cleaned everything myself!
Things I used:
First I cleaned my book shelf.
I arranged the books in the order of author's names, like first of all I put J. K Rowling, then David Williams, and others. I also put stickers on the side so that it's easy for me to find my books.
So this is how my book shelf looked like Before and After Cleaning:
Then I cleaned my drawer where I keep my things.
I took out all the things which I didn't need. And arranged the things properly that I needed.
I like the way my book shelf and drawer looks like now but I got so tired while doing all this!


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**The AHQA Quality Summit Hotel Block is Now Closed**
Hilton Baltimore (link is external)
401 W. Pratt Street
Baltimore, MD 20121
AHQA has secured a block of rooms at the Baltimore Hilton at a reduced rate of $189/night. Attendees are responsible for reserving their own rooms. Please click here (link is external)to make your reservation. The reduced rate is available until Friday, June 15 or until the room block is filled.
Hotel parking is $30/day for self parking and $42/day for valet. Check-in is at 4:00 pm.
How to Get There
(For a full list of directions please visit the Baltimore Hilton's website(link is external)).
Baltimore/Washington International
Take 195 West to 95 North. Take exit 53 (395 North). Follow for one half mile and turn right on Pratt Street. The hotel will be on the right.
- Distance from Hotel: 10 mi.
- Drive Time: 15 min.
Typical Minimum Charge
- Limousine 35.00 USD
- Super Shuttle 13.00 USD
- Rental Car Rates Vary
- Taxi 25.00
Washington National Airport
Take I-95N to Baltimore. Take Exit 53, I-395N downtown. Follow 4 lights to Lombard St.; make left. Go 3 blocks, make left onto S. Green St.; Go 1 block, turn Left onto Pratt St. Hotel is located on the right
- Distance from Hotel: 41 mi.
- Drive Time: 50 min.
Typical Minimum Charge
- Limousine 170.00 USD
- Super Shuttle 115.00 USD
- Rental Car Rates Vary
- Taxi 140.00 USD GET TURN-BY-TURN DIRECTIONS
Washington Dulles International Airport
VA-267E to Washington DC-Exit 18-I-495N to Baltimore. I-495N to Exit 27, I-95N to Baltimore. I-95N to Exit 53, I-395N downtown. 4 lights to Lombard St.; make left. Go 3 blocks make left onto S. Green St. One block, turn left onto Pratt St.
- Distance from Hotel: 70 mi.
- Drive Time: 75 min.
Typical Minimum Charge
- Limousine 130.00 USD
- Rental Car Rates Vary
- Taxi 100.00 USD
Local Attractions
The downtown location of the Baltimore Hilton makes it accessible to many of the attractions and historic sites of the city. According to its website the Baltimore Hilton
"holds a prime place in the Inner Harbor, a popular historic, cultural and business district. Our downtown hotel is next door to Camden Yards and across the street from M&T Stadium. We're also connected to the Baltimore Convention Center and a quick 15 minutes to BWI Airport. Restaurants, indoor/outdoor event spaces, fitness options - we have something for everyone."
Click here(link is external) for more information on local attractions.


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Any suggestions for managing the world's smallest kitchen? I have virtually no storage areas, and less than 2 square feet of counter (thus, trivets on the bedside table occasionally).
If it's your place, i.e. you own it, then build shelves on your wall. If you are renting you can build shelves and hang them on the walls. This would be between any cabnets and counter tops that you do have. Also there are small cabnets you can buy and put in the kitchen. Not much help, but been there done that.
I love this question. This is like cooking on a boat or a bus. Be brave. If you are living in say a studio apartment think of your living area as an extension of your kitchen. If you have a table in there, use that too. Move furniture aside and go to work. Think about a work surface that's mobile and on wheels so you can move it around where you need it.
magnet strips and metal siding on the walls. it allows you to stick anything - from spices, to pots and pan and utelsins on the walls.
I basically have the same issue, I've sort of solved it by using bookcases as shelves and I bought a rolling butcher block cart with storage underneith. I still need more space, but it works a lot better.
Pierino--it is exactly like cooking on a boat (but not moving)! I like your suggestion to see this as an adventure!
How much wall space do you have? I lived with a tiny kitchen in NYC...no cabinet space, but one decent-sized spare wall. I installed a peg board and kept nearly everything on it, from pots and pans to strainers and utensils. A hardware supply store will have everything you need. Domino Mag used to have a great tutorial online (RIP), these days I found an okay one from Apartment Therapy Kitchn:
Well here's an even better tutorial for installing a pegboard:
Wow, you've just described my kitchen! It's always a challenge, but something you learn to live with. Small kitchens are underrated, I think. Much more organized, and it trains you to buy less stuff.
I find my hanging pot rack saves a ton of space.
My flat-top range is like another counter top.
I really should do a big pantry/equipment purge someday soon, and get rid of the stuff I haven't used in over a year - that will open up more room I'm sure.
Also, if you don't mind looking at big ugly plastic storage bins in your dining room, they are very helpful for keeping dry goods stashed away.
My favorite space-saving trick is stackable cooling racks. Got them from Pampered Chef a few years ago. I stack them 4-high.
Love the shelving and peg rack idea if you've got a wall. Very Julia Child-esque! Wish I had a spare wall.
Love the peg-board--it looks so organized and serene. (And I miss Domino, too!)
I think it's funny that it appears that most of you folks who do the most and most exciting cooking also have small kitchens--I'm thinking that those glam, giant kitchens might lead to apathy? Or never get used?
I am thinking of tools and equipment that are multi-use: a large pyrex mixing bowl can also be a serving bowl can also go in the oven as a round baking dish (thrift stores sometimes have great pyrex). I love my salad spinner even though it is large, but the clear plastic bowl of it could be a salad server if needed. A large cast iron skillet can be turned upside down for cooking pizzas, maybe cookies if you use parchment paper, and it is perfect for anything that starts on the stove and ends in the oven (frittatas, etc.) Also, there are all sorts of "fold-up" tools, I've seen fold up colanders, cutting boards, etc. Maybe bowls and cups that stack or nest to maximize cabinet space. Last, what can you hang form the ceiling? Those wire basket thingies are good for lighter items, fruit, etc. but you can also install hooks (find the joists or look for bolts that are intended for ceiling mount) so you can hang pots, colanders, etc . . . Definitely make it an adventure!
When I nannied regularly, it used to KILL me that the people with the most well-stocked, luxurious kitchens were the people who never used them. Inwardly I was screaming "Whyyyyy?!!?!?" haha. I was going to suggest the pegboard and magnetic strips, too.. they saved my sanity in my previous tiny kitchen. And I have about eight of those stackable cooling racks.. they are AWESOME.
I used to have a book called "Cooking in a Small Kitchen" by Arthur Schwatrz. Unfortunately it's not in print, but you can usually find used copies on Amazon or half.com. It had lots of suggestions for tools, recommendations for the minimum you need, and vintage 1970's recipes that are easy to manage in a small kitchen (and would be fun to update if only I hadn't given away my copy ages ago...)
I have done some of my most interesting cooking in a boat kitchen with a small four burner hob with oven, a 4 cubic foot refrigerator, sink and 30 inches of counter space. It meant shopping more frequently and tight menu planning but it's doable. Sharp knives are a must. A good cutting surface is essential. The comments about multi purpose tools is important as well.
Our house has the largest kitchen we have had in the 39 years we have been married. And there is still not enough storage space or counter space! After cooking in the previously mentioned small galley while narrowboating in the UK, I am working on purging little used equipment and making sure my stock of staples is used before buying more.
In one small kitchen I had, I'd open a drawer and center a large cutting board over it and close the drawer to hold the board firm. It at least gave me a little more surface space to work on.
i lived in an apartment with a kitchen that had no counterspace at all and no drawers at all. i bought a butcher block and used my small dining table, and sometimes i would leave things to cool on the air conditioner (not because it was cold but because it was, simply, a surface).
and then i learned about ironing boards, and they changed my life. not only are they very portable and can they fold down flat, but they are designed to withstand heat, so they worked brilliantly for all those cookie sheets putted right out of the oven. they aren't stable enough to do prep like cutting (or mine wasn't, anyway), but i could lay out my ingredients on them. i covered mine with dishtowels to help keep them clean.
they also make for good buffet tables (pushed against a wall for stability) or low coffee tables with a tablecloth thrown over them.
Soozll's open drawer trick and vvvanessa's ironing board technique are genious!! I'm gonna have to steal those. Thanks for the great tips!! :)
i meant "pulled" from the oven, not "putted." sorry if that was confusing!
be organized. very, very organized. everything you have needs to have a place, and it needs to be in that place except when you're not using it. You MUST clean as you go. A good habit anyway, but you have to do or you'll end up with spills and tears. One pot meals are good, things like stews and soups (once did a Vietnamese hotpot in my 11' x 15' NYC walk-up). So are dishes that can be prepared ahead of time and served warm, but not piping hot. Indian food is great for that!


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Urban Sketchers Join First Friday Downtown August 2
FREE and open to the public, First Friday Downtown includes venues, galleries, and retailers throughout Downtown Colorado Springs, featuring artwork and cultural activities the first Friday of every month. The following are highlights for August 2, 2013, 5 p.m. to 8 p.m.
Urban Sketchers provide art in action experience
Artists will capture downtown's "vibe" with original artwork, created onsite at several locations throughout downtown. The sale of artwork will benefit the Colorado Springs Pioneers Museum, and be available for purchase at the museum store. Artists will be sketching along Tejon Street at Colorado Avenue, Pikes Peak Avenue, Kiowa Street and Bijou Street. This special addition to First Friday Downtown was developed as a partnership between Downtown Colorado Springs, Urban Sketchers Colorado Springs, and the Colorado Springs Pioneers Museum.
Street performances join August line up
As part of the Sidewalk Stage street performance program, three locations will entertain as part of this month's First Friday. Taiko drummers, Flies with Buffalo (music): on Colorado Avenue, between Nevada and Tejon;
Taiko Society (drumming): on Pikes Peak Avenue, between Cascade and Nevada; and Jason Bennett (guitar) and Hear Here Spoken Word, at the corner of Tejon and Bijou, from 4 to 7 p.m.
Art Openings
· Bridge Gallery Reincarnation, an exhibit by sculptor Carl Brandenburg and weaver Barbara Berger. Located at 218 W. Colorado Avenue.
· Boulder Street Gallery In the Clouds, a series of Sky Paintings by Deb Bartos. Located at 206 N. Tejon Street.
· Colorado Expressions gallery Experience the Days of Old, an exhibit of Navajo artwork by Allen Mose, the jewelry of his wife Annie and the handmade accessories of their son Ash. Mose will be on site to sign prints. Located at 122 E. Kiowa Street.
· Cottonwood on Tejon, Featured artists Suely Beccaro and Wilhelmina Steenberger.
· Dream Catchers Insta Art collaborative of photos. Also Paint & Sip class (RSVP required). Located at 103 S. Wahsatch, Suite 106.
· Gallery 113, new works by Mary Gorman and Karen Standridge and live music by Jason Bennett. Located at 113 N. Tejon Street.
· Kreuser Gallery and AHA Gallery, Tijah, an invitational show of 10 local artists with various works. 218 W. Colorado Avenue.
· The Modbo Fog of War (paintings) by Holly Parker Dearborn. Located at 17b and 17c E. Bijou, in the alley.
· S.P.Q.R. Works of Roy Linton. Located at 17b and 17c E. Bijou, in the alley.
· GOCA 121 Visual Environments, an exhibit with works by Colorado artists Pattie Lee Becker and Suchitra Mattai. 121 S. Tejon Street, Suite 100.
· The Colorado Native Live at Studio Bee free concert series will feature Mark's Midnight Carnival Show and The Mailbox Conspiracy. Located at Studio Bee at the Pikes Peak Center, 109 S. Cascade Ave. Doors open at 5:30, show at 6:30.
· Sonic Landscapes, public concert on an outdoor stage next to GOCA 121 and COPPeR. Located at 121 S. Tejon Street, Suite 100.
· Sparrow Hawk Cookware. Work by local artists Phil Vallejo, Diane Vallejo, and Langdon Foss. Plus, a selection of original animation art from Disney Studios, Warner Brothers and Hanna-Barbera. Located at 120 N. Tejon Street.
· Pikes Peak Chocolate & Ice Cream. Featuring hand crafted dimensional work by Mark Bowers. Located at 125 ½ N. Tejon Street.
All downtown metered parking is free every day after 6 p.m. Several City parking lots offer parking for only $1 in the afternoons and evening, including:
· Bijou/Cascade Parking Garage, 215 N. Cascade Avenue: $1 after 3 p.m. and all day weekends
· City Admin Building Parking Garage, 130 S. Nevada Street: $1 after 4 p.m. and all day weekends
· Kiowa Street Parking Garage, 127 E. Kiowa Street: $1 after 2 p.m. and all day weekends
Galleries located in the Depot Arts District provide parking in gallery parking lots.
About First Friday Downtown
First Friday Downtown features FREE monthly cultural events throughout downtown Colorado Springs. Galleries, museums, cultural organization, and retailers host art openings, artist receptions, performances, and more the first Friday of each month, from 5 - 8 p.m. Additional details including images is available at http://www.downtown80903.com/arts-and-culture/first-friday-downtown.
Downtown Colorado Springs is designated by the State of Colorado's Colorado Creative Industries as a Prospective Creative District. Included in the district are cultural landmarks, award-winning outdoor sculpture; independent galleries, and other one-of-a-kind cultural and entertainment opportunities. For more information visit www.downtowncs.com, or contact Downtown Colorado Springs at 719.886.0088.


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Host a Sweetest Game Day House Party
Gather your team for a great big game celebration featuring everybody's favorite ingredients for fun: SNICKERS and SKITTLES.
If you're selected and confirmed as a House Party host, your exclusive Party Pack will contain:
$15 Walmart Gift Card to purchase candy, napkins, cups and plates for Party Day
Table football fooseball game
And more!
Apply for this opportunity on the House Party website today.
This offer is only valid for residents of:
- United States


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So I picked these up awhile back and decided to finally try them out. Orly not only decided to release two flakie top coats, but made them matte! I swatched them quickly over Sally Hansen Black Out so that the colors would show through nicely.
Green Flakie Top Coat is jam packed with shades of blue flakes. I used two coats, it gets really nice coverage. I didn't use a top coat over it, so you can see how well the matte effect works. The flakes are a nice size and aren't too thick, making them easy to layer.
Pink Flakie Top Coat has a variety of colors and shades of flakes. Two coats of this has a little less coverage than the Green Flakie, but its still a beautiful color. The flakes also seems to be a bit smaller in this color, but just as easy to handle.


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Summary | Excerpt | Reviews | Readalikes | Genres & Themes | Author Bio
Everything You Need to Know to Stay Healthy in the Decade before Menopause
You're in the prime of life. As far as you know, menopause could be years away. So why is your body sending you such weird messages? You could be in perimenopause--the change that precedes the change by as many as ten years.
"What's happening to me?"
You're in the prime of life. As far as you know, menopause could be years away. So why is your body sending you such weird messages?
If even one of these sounds familiar, you could be in perimenopause--the change that precedes the change by as many as ten years. Has your doctor mentioned it? Shockingly, many still don't. But Dr. Laura Corio has "the talk" with every one of her patients when they reach age 35. And now, in this lively and solution-packed book, she shares her experience helping thousands of women navigate these years in peak physical and emotional health.
You'll learn the how's--up-to-the-minute options for getting your symptoms under control. You'll also learn the fascinating why's--from the subtle hormonal shifts that affect every part of your body to the impact of stress and diet. You'll get precise details on:
Above all, The Change Before the Change puts you back in charge, with the confidence and resources you need to make the right decisions during these pivotal years.
There was a huge snowstorm raging outside, and we'd been receiving cancellation calls all day. So I was surprised to open the door to one of our examining rooms and find Marian perched on the end of the table. Marian started seeing me twelve years ago, when she was single and living in the city. A husband and two children later, she now lived in the suburbs but still remained a loyal patient.
"Why didn't you reschedule?" I asked. "You took your life in your hands driving all the way in from Long Island."
"I couldn't wait another day, Dr. Corio," she replied.
"What's wrong?"
"What isn't? I feel like my whole body is falling apart--in fact, it doesn't even feel like my own body anymore!" Marian went on to list a host of symptoms: migraines, hot flashes, insomnia, dry itchy skin, urinary tract infections, irregular periods, and decreased libido. "Poor Skip," she continued, referring to her sweetheart of a husband. "My moods have been all over the place...
If you liked The Change Before The Change, try these:
Instead of dreading menopause as a time of loss, women will hear the real message their bodies are sending--that this is a time of personal empowerment and positive energy . . . and a time for women to break free and thrive.
Uncommon Type
by Tom Hanks
Surprising, intelligent, heartwarming, and a must-have for Tom Hanks fans!
Solve this clue:
and be entered to win..
Visitors can view some of BookBrowse for free. Full access is for members only.
Your guide toexceptional books
BookBrowse seeks out and recommends books that we believe to be best in class. Books that will whisk you to faraway places and times, that will expand your mind and challenge you -- the kinds of books you just can't wait to tell your friends about.


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Wednesday, March 14, 2012
My Style: Edgy, Daytime Chic
Recently I went on a whirlwind European vacation and made it into my own personal fashion show. Kind of how I do everyday with my life. A pleasant daytime stroll in Zurich, Switzerland turned into a city wide photo shoot.
Vince Camuto Sunglasses, Club Monoco top, Leather Bomber (Loehmanns), DVF lace motorcycle gloves, Adrienne Landau fur scarf, Armitron watch and a Helmut Lang blazer.
Posted by Amy Salinger at 7:39 AM 1 comments
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Various TVpad firmware upgrade packages to the latest firmware version 3.26 has been released and can be found below. Go to Settings icon (grey icon) and then system info icon (last icon) to check your current firmware version. Depending on your current hardware model and firmware version, download the correct firmware upgrade package.
Go to this page for the TVpad3 promotion packages: tvpadm233.blogspot.com/2013/11/tvpad-3-discount-deals.html
Supported hardware models: TVpad M120-3201, M120-3301, M121-3311, M121S-3311
Supported firmware versions: 2.62, 2.70, 2.72 (for M120 and M121 users)
3.06, 3.12, 3.14 (for M121S users)
TVpad firmware upgrade packages to 3.26 download
Instructions to upgrade your current firmware version to firmware version 3.26:
Note: you will need to install the apps again after the firmware upgrade to 3.26. All the apps can be found in the "TVpad store" inside "App Manager".
1. First, download the correct firmware upgrade package depending on your current hardware model and firmware version.
2. Then, put the downloaded upgrade package (.tar file) in an USB-stick (don't put it in a folder).
3. Now, start your TVpad and connect your USB-stick with your TVpad. Temporary, remove the micro-sd card from your TVpad (only if your TVpad model has a micro-sd card slot)
4. Go to settings (grey icon on the left side), then go to the green upgrade icon (with 2 arrows in the icon). After that, allow it to update to firmware 3.26. It takes about 5-10 minutes to update the firmware and it will automatically reboot your TVpad after that. After the firmware upgrade has been completed, it will take about 5-10 minutes to start-up for the first time.
5. After the firmware upgrade to 3.26, you need to install the apps again in the "TVpad store" inside "App Manager". Take a look at the search app (应用搜索器) and install it (more apps can be found in this search app, including 粵海寬頻, 粵海時移, HITV and 港粤快看).
After opening the search app, click on the "OK" button to see all the apps available.
Then, click on the apps (in Chinese words) you would like to download. The downloaded apps are stored in your micro-sd card.
Then go to the "Others" tab inside "App manager". In this tab, you can install the apps you have downloaded..
Note: If you don't see any apps in the "TVpad store" inside "App Manager" after the firmware upgrade, first find out whether the user icon on the bottom (right) is green or grey. If it's grey or if you are getting "the user does not exist message", contact the customer service about your problem and include your MAC address.


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Raster clipart rock
Raster coloring rock - 1182х1715
Raster coloring rock - 567х822
Apply raster clipart rock
Picture of a multi-faceted rock transmits the characteristic form of the mark in the rock, for the perception of hand-drawn image. In the life of this contrast is not practical to separate but knowledgeable people there is a similar way, because the way it should be.
Raster picture makes it possible to quickly decorate the different items or files. Create backgrounds. Combination of several clipart, create a unique decorative elements. In the bitmap editor can always adjust the color (example: darken, highlight, pasteurized, substitute). Images can be transformed into a kind of a unique pattern (engraving, watercolor, mosaic, etc.). For this purpose, such as applying different filters. You can create brushes for Photoshop. Increasing the transparency of clipart, it fits easily into the interior. In general, there is no fantasy aisle.
Artwork created by coloring colorings, which you can download on the site. Clipart may not always reflect the real color of the object depicted in it, the artist is given full opportunity for fantasy. Artists to design clipart are mostly children.


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In the resolution of appointment, Mr. Danzansky stated he, "desires to publish and make more publicly available the collection of artifacts and information in his possession and provide a more open and accessible vehicle and agent for the maintenance and dissemination of information concerning (the) Diplomats."
|Resolution of Appointment and Agreement
|The only competitive trophy awarded to the franchise in their history, from the 1978 Presidents Cup, Seoul, Korea.
|Johan Cruyff Match Jersey.
|Circa 1977/1978 team warm up suit.
1977 D.C. City Council Resolution honoring the Diplomats.
|Press Archive.


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A while back I wrote about my former classmate Timmy. While he was in India, he was asked to speak at a TEDx Talk. I've imbedded the video below. He talks about making connections and his experiences in finding ways to be connected to people. Through his travels, he was able to get a sense of what it feels like to live in the moment, where connections truly take place, but he was able to synthesize the information into a set of guidelines he can bring back to his life in the Western world, and he shares them with us, here.
New Year
4 days ago


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The club is -- The Global Information Network (GIN) and it is a private exclusive member-only club styled after the Secret Clubs of the World. It is a global association of individuals dedicated to achieving their goals:
- Never Having to Ask the Price of What your Want -- Financial independence
- Law of Attraction and Wealth creation
- Dynamic health
- And happiness, Joy and emotional well-being
The goal of the secret club is to provide a safe haven where members can build mutually beneficial long-term relationships to help one another better achieve each of their goals, desires and dreams. The belief is that this rare and unique ability to associate with people from around the world allows each member special advantages that they would not have as isolated individuals.GIN is a global group of like minded highly influential, affluent, and freedom-orientated people from various business, social and economic sectors.
Those joining the secret club share a desire to help other members share knowledge, connections, and expertise, and mentor those members who are serious about taking charge of their own destinies, achieving wealth, gaining financial freedom, experiencing dynamic health, and reaching high levels of overall emotional well-being. Members include highly accomplished leaders from a large variety of industrial, financial, social and political sectors. Many members have major professional accomplishments, academic accolades and worldwide reputations and achievements.
The GIN membership list is confidential.
This secret Club provides its members with exclusive, confidential, never before released information regarding:
- Steps: The correct Way to use The Law of Attraction
- Money Making Opportunities
- Investing
- Worldwide Real Estate Opportunities
- Training in Stocks, Bonds and Trading
- Sharing knowledge in Commodities, Worldwide Currencies
- Off-shore Banking- where, how and who to trust
- Asset Protection global
- Natural Remedies and Cures for Illnesses
- And much more
International Membership is by invitation only. All members of GIN are and will always remain anonymous to the general public. GIN is a Multi-Form Foundation organized in the country of Nevis.
Check out these Links below:
Global Information Network
Bilderberg Secret Club
The Illuminati secret
Your Wish Is Your Command CD's
Want to be Invited to join Global Information Network? Our affiliate code is money8 or 1512126. We'll help you become financially free. Want to join for free? Just join as an affiliate and get the training today!