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While the molecular mechanism of membrane fusion by the influenza virus hemagglutinin (HA) protein has been studied extensively in vitro, the role of acid-dependent HA protein activation in virus replication, pathogenesis, and transmission in vivo has not been characterized. To investigate the biological significance of the pH of activation of the HA protein, we compared the properties of four recombinant viruses with altered HA protein acid stability to those of wild-type influenza virus A/chicken/Vietnam/C58/04 (H5N1) in vitro and in mallards. Membrane fusion by wild-type virus was activated at pH 5.9. Wild-type virus had a calculated environmental persistence of 62 days and caused extensive morbidity, mortality, shedding, and transmission in mallards. An N114K mutation that increased the pH of HA activation by 0.5 unit resulted in decreased replication, genetic stability, and environmental stability. Changes of +0.4 and −0.5 unit in the pH of activation by Y23H and K58I mutations, respectively, reduced weight loss, mortality, shedding, and transmission in mallards. An H24Q mutation that decreased the pH of activation by 0.3 unit resulted in weight loss, mortality, clinical symptoms, and shedding similar to those of the wild type. However, the HA-H241Q virus was shed more extensively into drinking water and persisted longer in the environment. The pH of activation of the H5 HA protein plays a key role in the propagation of H5N1 influenza viruses in ducks and may be a novel molecular factor in the ecology of influenza viruses. The data also demonstrate that H5N1 neuraminidase activity increases the pH of activation of the HA protein in vitro.
Highly pathogenic H5N1 influenza viruses were transmitted to humans in 1997 in Southeast Asia (7) and have subsequently spread across Asia, Europe, and Africa (53). Millions of poultry have been culled to control outbreaks (19), and more than 250 human lives have been lost (http://www.who.int/csr/disease/avian_influenza/en/ ). These viruses appear currently to lack the molecular properties required for sustained transmission among humans. There is an urgent need to understand the molecular properties that contribute to the transmission and host range of these viruses for their effective surveillance and containment.
The transmissibility and pathogenicity of influenza A viruses, including the H5N1 subtype, in avian and mammalian species are determined by both viral and host factors (8, 39). One key factor is the multifunctional hemagglutinin (HA) protein. During viral entry, the HA protein binds to sialic acid-containing receptors on host cells; the virus then undergoes endocytosis, and its HA protein is activated at a low pH to cause the fusion of the viral and endosomal membranes (11, 41). The host range of influenza A viruses depends in large part on the receptor specificity of the HA protein. Avian influenza viruses generally bind to α(2,3) sialosides with greater affinity, while human influenza viruses usually bind to α(2,6) sialosides with greater affinity (4, 37). The receptor binding affinities and specificities of HA proteins also depend on internal linkages and modifications of inner oligosaccharides, and glycan microarray profiling has revealed differences in receptor binding between seasonal human influenza viruses and H5N1 viruses (23, 45, 46). Thus, the natural distribution of various sialosides in different tissues of different species helps to determine both tissue tropism and species specificity (31, 40, 50, 58). The posttranslational cleavability of the HA0 precursor protein into the fusion-capable HA1-HA2 complex is a critical determinant of the virulence of influenza viruses (16, 22, 55). The presence of a polybasic cleavage site in H5 and H7 influenza viruses allows HA protein cleavage in the trans-Golgi network by ubiquitous furin-like enzymes and is a marker of high pathogenicity (12, 38, 55).
During entry into host cells, influenza viruses are exposed to increasingly lower pHs until a threshold is reached at which HA protein trimers undergo irreversible conformational changes that promote membrane fusion (11, 41). Threshold pH values differ among influenza viruses, and a change in the pH of fusion of the HA protein can help influenza viruses to adapt to different cell lines (5, 25) and host species (13) or to the higher endosomal pH induced by high concentrations of the antiviral drug amantadine (6, 9, 42-44). In general, a high pH of HA protein activation could result in influenza virus inactivation in the environment or during transport to the cell surface for intracellularly cleaved HA proteins (2, 44). On the other hand, a low pH of HA protein activation could result in degradation in the lysosome as the pH of the endocytic pathway decreases from early endosomes to late endosomes to lysosomes (61). Therefore, for efficient propagation within a biological host and ecological niche, an influenza virus may have an optimal range of pHs of activation for the HA protein. Moreover, the optimal activation pH may change upon introduction of an influenza virus into a new host species or environment.
Aquatic birds are a natural reservoir of influenza viruses, but surprisingly little is known about the molecular basis of influenza virus propagation in these species. To test the hypothesis that the pH of activation of the HA protein contributes to the pathogenicity and transmissibility of H5N1 influenza viruses in the mallard, a prototypic aquatic bird, we previously generated four recombinant H5N1 viruses containing mutations that altered the acid stability of the HA protein without changing its level of expression, cleavage, receptor binding, or membrane fusion efficiency (36). Two of the mutations increased the pH of membrane fusion of the H5N1 HA protein (Y231H and N1142K), and the other two mutations reduced the pH of fusion (H241Q and K582I). HA1 mutations Y231H and H241Q (H5 numbering with subscripts denoting HA1 and HA2 subunits) are located in the fusion peptide pocket and were originally chosen because of their presence in H1 and H9 subtypes, respectively. The K582I mutation in the A-helix of HA2 was chosen because it decreases the pH of membrane fusion of the H3 HA protein by 0.7 unit (44). The N1142K mutation in the fusion peptide pocket was chosen because it increases the pH of membrane fusion by approximately 0.5 unit in H3 and H7 subtypes (6). Here we measured the effects of the H5 HA protein mutations on virus replication in vitro, on genetic stability after repeated passage in eggs, and on environmental stability. Mallards were inoculated with the recombinant viruses and were housed with contact ducks in order to determine the effects of the mutations on virus shedding, pathogenesis, and transmissibility. An H241Q mutation in the HA protein was found to decrease the pH of activation by 0.3 pH unit, to increase the titers of infectious virus recovered from ducks' water dishes, and to prolong the persistence of infectious virus in the environment. In general, changes in the acid stability of the HA protein were found to alter H5N1 influenza virus replication, pathogenicity, and transmissibility.
Viruses, plasmids, and cell culture.Recombinant viruses and plasmids containing HA protein mutations Y231H, H241Q, K582I, and N1142K were generated previously (36). All experiments using H5N1 influenza viruses were performed in a USDA-approved biosafety level 3+ containment facility. Monolayer cultures of Vero cells (ATCC CCL-81) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% glutamine, 1% penicillin, and 1% streptomycin. Monolayers of Madin-Darby canine kidney (MDCK) cells (ATCC CCL-34) were grown in minimum essential medium (MEM) supplemented with 10% fetal bovine serum, 1% glutamine, 1% penicillin, and 1% streptomycin.
Virus growth kinetics.Single-step growth curves in MDCK cells were determined for each of the recombinant viruses. Confluent monolayers were infected at a multiplicity of infection (MOI) of ∼3 PFU per cell. After 1 h of incubation, cells were first washed with a 0.9% aqueous NaCl solution (pH 2.2) to remove any free infectious virus particles and were then washed twice with phosphate-buffered saline (PBS) to adjust the pH. Cells were incubated at 37°C in MEM (containing 4% bovine serum albumin and 1% glutamine). Supernatants were collected 2, 4, 6, 8, and 10 h postinfection and were stored at −70°C. To determine multiple-step growth kinetics, MDCK cells were infected at an MOI of ∼0.01 PFU/cell. After 1 h of incubation, cells were washed twice with PBS and were incubated at 37°C in MEM (containing 4% bovine serum albumin and 1% glutamine). Supernatants were collected 12, 24, 36, 48, 60, and 72 h postinfection and were stored at −70°C. The virus was titrated as described previously (60). Briefly, confluent MDCK cells were incubated for 1 h at 37°C with 10-fold serial dilutions of virus in 1 ml infection medium. The cells were then washed and overlaid with freshly prepared MEM containing 0.3% bovine serum albumin and 0.9% Bacto agar. After incubation at 37°C for 3 days, plaques were visualized by using a 0.1% crystal violet solution containing 10% formaldehyde.
Genetic stability.The H5N1 influenza viruses were serially passaged in 10-day-old embryonated chicken eggs to assess the genetic stability of the introduced mutations. Eggs were infected with 1 HA unit of sequence-confirmed virus. Allantoic fluid was collected, and the HA titer was measured to determine the dilution for subsequent passage of the virus. RNA was extracted and sequenced as described above.
Environmental stability.Stocks of recombinant viruses were diluted 1:50 in distilled water (pH 7.4) containing 2 mM HEPES buffer. Aliquots were incubated at 28°C (the approximate environmental temperature in Louisiana during the summer, allowing comparison with data from similar studies) (2). Aliquots were removed daily for 8 days, and their titers measured by plaque assay were compared to the initial virus titer. The sequential data were log10 transformed and analyzed by linear regression using GraphPad Prism software (GraphPad Software, La Jolla, CA). The gradient from this model was then used to calculate the estimated persistence of 1 × 106 PFU/ml of recombinant virus and the time required to reduce the infectivity of the initial inoculum by 90% (1 log10). Differences in the linear regression models were measured by using GraphPad Prism software.
Inoculation and transmission studies of mallards.Groups of three 4-week-old mallards (Anas platyrhynchos) were inoculated via intranasal, intraocular, and intratracheal instillation of ∼106 50% egg infective doses (EID50) of virus in a 1-ml volume, as described previously (21). Two uninoculated contact ducks were placed in the cage with the inoculated ducks 24 h postinoculation (p.i.), and shared a common food and water source. Birds were weighed and observed daily for signs of morbidity or mortality over a period of 14 days. Birds that did not eat or drink on their own due to severe disease signs were euthanized, and their deaths were recorded on the following day of observation. Tracheal and cloacal swabs were collected from all ducks on days 3, 5, 7, and 10 p.i., and 0.5 ml of drinking water was sampled on days 1, 3, 5, 7, and 10 p.i. Influenza virus was detected by virus isolation in 10-day-old embryonated chicken eggs as previously described (14, 47). The virus was titrated in positive samples by calculating the EID50, using the method of Reed and Muench (35); the lower limit of quantification was 0.75 log10 EID50/ml. Swab samples with detectable influenza virus but titers below the limit of quantification were reported as having a titer of <101 EID50/ml. All data shown were derived from two separate experiments. All animal experiments were approved by the Animal Care and Use Committee of St. Jude Children's Research Hospital (Memphis, TN) and were performed in compliance with relevant institutional policies, the Association for the Accreditation of Laboratory Animal Care guidelines, the National Institutes of Health regulations, and local, state, and federal laws.
Transient expression of HA and NA proteins.Monolayers of Vero cells in 6-well dishes (85 to 95% confluence) were transiently transfected with 1 μg of pCAGGS A/chicken/Vietnam/C58/04 HA DNA by using the Lipofectamine Plus expression system (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Transfected Vero cells were incubated for 4 h at 37°C. DMEM (containing 10% fetal bovine serum and 1% glutamine) was then added to cells, and cells were incubated for 16 h at 37°C. Cells were then treated as indicated for each experiment. Neuraminidase (NA) protein was expressed by using 0.1 to 1.0 μg of the pCAGGS A/chicken/Vietnam/C58/04 NA plasmid.
Syncytium assay.Monolayers of Vero cells grown in 6-well plates were transfected with 1.0 μg pCAGGS HA as described above or were infected with recombinant virus at an MOI of ∼3 PFU per cell. At 16 h posttransfection or 6 h postinfection, cell monolayers were overlaid for 5 min with phosphate-buffered saline with magnesium and calcium (PBS+) that was adjusted to the reported pH with a 0.1 pH unit resolution using 0.1 M citric acid. Cells were neutralized by using DMEM (containing 10% fetal bovine serum and 1% glutamine) and were incubated at 37°C for 2 h. Samples were fixed and stained with a Hema 3 stat pack staining kit (Fisher) according to the manufacturer's instructions. Representative microscopic fields were captured with a Nikon D70 digital camera attached to a Nikon Eclipse TS100 inverted microscope (26).
NA activity assay and NA inhibition.A modified fluorometric assay was used to determine the enzymatic activity of the NA protein present in transfected cell lysates with the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNANA; Sigma, St. Louis, MO) (15, 34, 59). The fluorescence of the released 4-methylumbelliferone was measured in a Fluoroskan II spectrophotometer (Labsystems, Helsinki, Finland) using excitation and emission wavelengths of 355 and 460 nm, respectively. The enzymatic activity of NA protein was standardized to 0.1 mg total protein by using a bicinchoninic acid assay (Sigma, St. Louis, MO) and was expressed as the quantity of substrate (in picomoles) converted during a 30-min incubation at 37°C. The NA inhibitor oseltamivir carboxylate ([3R,4R,5S]-4-acetamido-5-amino-3-[1-ethylpropoxy]-1-cyclohexene-1-carboxylic acid) was provided by Hoffmann-La Roche, Ltd. (Basel, Switzerland). The compound was dissolved in distilled water, and aliquots were stored at −20°C until use. NA activity was inhibited by using a 4 μM concentration of oseltamivir carboxylate added immediately posttransfection or 1 h before the assay. The effect of an NA protein inhibitor on the pH of fusion was determined by performing a syncytium formation assay in parallel.
The HA protein mutations have little effect on the in vitro replication kinetics of recombinant H5N1 influenza viruses.In a previous study, we identified four mutant H5 HA proteins whose pH values of membrane fusion differed from that of wild-type HA protein when expressed from transiently transfected plasmid DNA (36). Here we determined whether the HA protein mutations affected the in vitro replication kinetics of recombinant H5N1 influenza viruses by generating single-step and multiple-step growth curves. Single-step growth curves showed that mutant and wild-type viruses grew at similar rates over the 10-h time course (Fig. 1A). In multiple-step growth curves, viruses containing the HA protein mutations Y231H, H241Q, and K582I had replication rates similar to that of wild-type virus (Fig. 1B). Titers of the virus containing an N1142K mutation in the HA protein were similar to those of wild-type virus at the 12-h time point but were later reduced by 1 to 3 log10 units.
The HA protein mutations alter the pH of membrane fusion in vitro.The cell surface expression, cleavage, receptor binding affinities, and membrane fusion efficiencies of the mutant HA proteins in Vero cells were previously found to be similar to those of wild-type virus (36). Moreover, infection of DF-1 primary chicken embryonic fibroblasts with the viruses resulted in HA protein properties similar to those found when Vero cells were infected with the viruses. To determine the effects of the mutations on the pH of membrane fusion, monolayers of MDCK cells were first infected with recombinant H5N1 influenza viruses at an MOI of ∼3 PFU/cell and then exposed to PBS solutions of varying pHs, at a resolution of 0.1 unit. The highest pH at which cell-cell membrane fusion was induced in cells infected with wild-type virus was 5.9 (Fig. 2). The H241Q and K582I mutations reduced the pH of membrane fusion to 5.6 and 5.4, respectively, while the Y231H and N1142K mutations increased the pH of membrane fusion to 6.3 and 6.4, respectively. The N1142K mutation, which increased the pH of HA activation by 0.5 pH unit, resulted in decreased virus fitness over several cycles of replication in vitro, while the other mutations did not alter in vitro replication kinetics.
The N1142K mutation is genetically unstable over multiple passages in eggs.To test the genetic stability of the HA protein mutations, wild-type and mutant viruses were passaged 10 times in 10-day-old embryonated chicken eggs. The sequence identity of each of the passage 1 (P1) recombinant viruses had been confirmed previously (36). Purified viral RNA sampled from allantoic fluid at P5 and P10 was sequenced. In parallel, syncytium formation assays were performed using Vero cells infected with P10 viruses to determine whether repeated passage in eggs resulted in any mutations that might alter the acid stability of the viral HA proteins (Table 1). Wild-type virus and viruses containing the HA protein mutation H241Q or K582I showed no additional mutations over the course of 10 passages and no change in the pH of membrane fusion. The virus containing the Y231H mutation maintained the mutation for at least 5 passages in eggs and acquired an additional HA protein mutation, R2281I, between P5 and P10. Residue R228 (H5 numbering) is located in the receptor-binding pocket of the HA1 subunit with its side chain facing away from the pocket (46, 57) such that the R2281I mutation may enhance receptor binding in eggs (56). Despite the extra R2281I mutation, the P10 virus caused membrane fusion at a pH of 6.3, as did P1 Y231H virus without the additional R2281I mutation. The virus containing the N1142K mutation in the HA protein was the only recombinant virus whose pH of membrane fusion was altered at P10 from that for the P1 stock virus, a decrease from pH 6.4 to 6.1 (Table 1). Both P5 and P10 K1142N viruses showed reversion mutations, demonstrating that the N1142K mutation was not genetically stable and was selected against within 5 passages.
Changes in the pH of activation of the HA protein can alter the environmental stability of H5N1 influenza viruses.The environmental stability of highly pathogenic H5N1 isolates has been found to be lower than that of lower-pathogenicity viruses (1, 2). To determine whether changes in the pH of fusion of the HA protein alter the environmental stability of H5N1 viruses, we incubated the viruses in the present study at 28°C for 8 days and measured the virus titer as a function of time by a plaque assay. Data from each series were plotted, and the gradient of virus degradation was calculated by linear regression analysis (Table 2). The wild-type virus and the virus containing a Y231H mutation in the HA protein showed similar rates of titer reduction (1 log10 unit every 10 days), a rate of degradation that matches those of other highly pathogenic H5N1 isolates (2). This result suggests that changes in the pH of fusion as great as +0.4 pH unit can be tolerated without a loss in environmental stability. Viruses containing the H241Q or K582I mutation, both of which promoted membrane fusion at lower pH values than wild-type virus, were calculated to lose 1 log10 unit in their titers every 13 days. Thus, the two mutant viruses with lower pH values of activation retained infectivity longer than the wild-type virus. The virus containing an N1142K mutation rapidly lost infectivity in the environmental stability experiment, losing 1 log10 unit in its titer approximately every 2 days (Table 2). Therefore, an increase in the pH of HA activation to 6.4 due to the N1142K mutation resulted in greatly reduced environmental stability, and a decrease in the pH of activation of the HA protein to 5.6 or 5.4 due to the H241Q or K582I mutation, respectively, moderately increased environmental stability.
The pH of activation of the HA protein contributes to the pathogenicity and transmissibility of H5N1 viruses in mallards.To measure the biological properties of the mutant viruses in mallards, we inoculated duplicate groups of three animals and introduced two contact animals into the cage of each group after 1 day. The wild-type and H241Q viruses induced considerable weight loss in both inoculated and contact animals (Fig. 3) and caused death in 60% and 70% of animals, respectively (Table 3). In contrast, the Y231H and K582I viruses did not induce weight loss or death in either inoculated or contact animals. Moreover, the Y231H virus caused only cloudy eyes for 50% of the inoculated ducks, while the K582I virus caused cloudy eyes only for one contact duck. While the virus containing an N1142K mutation in the HA protein did not induce weight loss or death in inoculated ducks, contact animals in this group unexpectedly showed weight loss after 4 days, and three of the four contact animals died. Neurological signs were observed in these contact animals, whereas none were observed in the inoculated group. Because of these unexpected findings, we sequenced viral RNA isolated from positive swabs from surviving inoculated and contact birds on days 7 and 10. In all cases, the N1142K mutation had reverted to the wild-type N1142, as it had after serial passage in eggs (Table 1). This reversion offers the most plausible explanation for the increased transmissibility and pathogenicity in contact birds in the N1142K group. The reversion mutation also explains the greater weight loss, morbidity, and mortality in the contact birds than in the infected birds in this group. No other reversion mutations were sequenced from swabs of contact ducks infected with the other viruses, including the H241Q virus.
To assess replication and transmission potential, titers of virus shed from the trachea and cloaca were measured (Table 4). Inoculated birds in all groups shed virus on day 3; therefore, it is clear that all of the viruses were capable of initial infection and replication. However, even at this early time point, the viruses containing HA protein mutation Y231H or K582I productively infected fewer ducks than wild-type and H241Q viruses. Wild-type and H241Q viruses were shed at similar levels on days 3 and 5. On day 7, wild-type virus was not shed, whereas the H241Q virus continued to be shed. All contact birds in the wild-type and HA241Q groups were shedding virus by day 3 p.i. (100% transmission). All contact birds in the H241Q group succumbed to infection, whereas only half of the contact birds in the wild-type group died. The Y231H virus was not detected in any contact birds throughout the experiment, showing that the mutation results in attenuated transmission compared to that of the wild-type virus. Five days p.i., the K582I virus was detected in one inoculated bird and one contact bird, showing that its fitness and transmissibility were lower than that of the wild-type virus. The N1142K virus showed inconsistent shedding in inoculated birds. Inoculated birds shed virus on day 3 but not on day 5, yet some birds again shed virus on days 7 and 10. This result suggests that transmission to contact birds was mediated by the reverted K1142N virus, which was then transmitted back to inoculated ducks before being detected on days 7 and 10. On day 3 p.i., tracheal shedding was generally observed more often and at higher titers than cloacal shedding, consistent with previous work (48). The H241Q and K582I mutations did not appear to increase cloacal virus shedding; thus, small decreases in the pH of activation (and inactivation) of the HA protein may be insufficient to enhance virus replication in the low-pH environment of the duck digestive tract (49).
We also investigated shedding of the recombinant viruses by titrating virus in the ducks' water dishes. Wild-type virus was detected on days 1, 3, and 5 p.i. and had a peak titer of 3.25 log10 EID50 on day 3 (Table 5). No Y231H virus was detected on any day, consistent with low shedding of this virus on day 3 and none on days 5, 7, and 10 p.i. The K582I virus titer in the water dishes was comparable to that of the wild-type virus on days 1 and 3 but was subsequently undetectable, consistent with the pattern of virus shedding from the tracheae and cloacae of ducks (Table 4). The presence of the N1142K virus in water dishes on days 3 and 5 but not on day 1 is consistent with low-level shedding until after reversion. Higher titers of the H241Q virus than of wild-type virus were detected in the water dishes on days 1 through 7, consistent with this mutant's greater environmental stability and lethality in contact ducks. Overall, our results show that the reduction of the pH of membrane fusion for the virus containing an H241Q HA mutation enhances two properties that could promote H5N1 virus transmission in aquatic birds: shedding of virus into water and persistence of virus infectivity in water.
The properties of H5N1 influenza viruses reported in Fig. 1 to 3 and Tables 1 to 5 are summarized in Table 6.
NA activity promotes pH-mediated membrane fusion induced by the HA protein.The highest pH at which wild-type virus caused membrane fusion was 5.9 (Fig. 2); however, we previously found that wild-type HA protein expressed from transiently transfected plasmid DNA caused membrane fusion only when the pH was decreased to 5.5 (36). The occurrence of this change in the context of virus infection (with expression of all viral proteins) in the present study suggested that one or more of the other viral proteins promote acid-induced activation of the H5N1 HA protein. Previous studies have shown that the NA protein facilitates the entry of H3N2 influenza viruses (28, 32). To determine whether NA protein expression increases the pH of membrane fusion by the HA protein, we transfected Vero cells with the pCAGGS HA wild-type plasmid in the presence and absence of cotransfection with the pCAGGS NA wild-type plasmid. Titration showed that 0.1 μg of plasmid DNA produced neuraminidase activity similar to that in 10 μl of allantoic fluid containing virus (data not shown); therefore, a 1:0.1-μg ratio of HA to NA was used in all follow-up experiments. Transfected cells expressing the wild-type H5N1 HA and NA surface proteins showed syncytium formation at pH 5.9, the same pH as that for wild-type virus (Fig. 4A). Having established that expression of the NA protein accounted for the observed increase in the pH of HA-mediated membrane fusion, we next examined whether NA enzymatic activity was responsible for the increase. NA enzymatic activity was eliminated in Vero cells cotransfected with pCAGGS HA and NA plasmids by treatment with oseltamivir carboxylate (3, 51) (Fig. 4B). The syncytium formation assay was repeated using cells coexpressing the HA and NA proteins in the presence of oseltamivir carboxylate. When NA enzymatic activity was inhibited by the drug, the pH of HA-mediated membrane fusion decreased to pH 5.5, the same value observed in cells expressing HA protein alone (Fig. 4A). These results are consistent with the promotion of H5 HA-mediated membrane fusion by N1 neuraminidase activity.
To investigate how the pH of activation of the HA protein influences the in vitro and in vivo properties of influenza viruses, we compared four recombinant viruses with altered pH-dependent HA protein stability to wild-type A/chicken/Vietnam/C58/04 (H5N1) virus. An N1142K mutation in the HA2 fusion peptide pocket region increased the activation pH of the HA protein from 5.9 to 6.4, allowing activation under mildly acidic conditions. This mutation dramatically reduced the fitness of the virus in three ways: (i) multiple-step replication in vitro was reduced by a factor greater than 10; (ii) infectivity in the environment decreased four times as rapidly as that of wild-type virus; and (iii) the virus reverted to the wild-type sequence within 5 passages in chicken eggs and after inoculation in mallards. The N1142K mutation may increase the pH of activation of the HA protein above the threshold pH at which a significant portion of intracellularly cleaved HA trimers become prematurely triggered, and inactivated, during transport to the cell surface (44). The HA protein mutations Y231H and K582I changed the activation pH of the HA protein to 6.3 and 5.4, respectively. While these two mutations had opposite effects on the activation pH, the recombinant viruses bearing the mutations had similar phenotypes. Despite in vitro replication rates similar to that of wild-type virus, the viruses bearing a Y231H or K582I mutation did not induce weight loss, neurological signs, or mortality in mallards, were not efficiently transmitted, and were shed significantly less. Overall, the data show that efficient and sustainable infection of mallards by H5N1 influenza virus is not supported by HA protein activation pH values less than 5.5 or greater than 6.2.
The results of experiments with the virus bearing an HA-H241Q mutation suggest that robust infection in mallards is supported by activation pH values between 5.6 and 5.9. The data also raise the possibility that natural mutations that slightly reduce the pH of activation of the HA protein could increase the transmission of H5N1 influenza viruses among mallards. The wild-type virus and the HA-H241Q virus had similar replication kinetics in vitro and induced similar weight loss, mortality, clinical signs, and shedding in mallards, but higher titers of the H241Q virus were found in the ducks' water dishes, and the H241Q virus retained infectivity ∼20% longer than wild-type virus in an environmental stability experiment. The fact that all of the contact ducks succumbed to infection with transmitted H241Q virus while only half died from transmitted wild-type virus also suggests that contact ducks were exposed to a larger inoculum of the H241Q virus.
Our results demonstrate that the pH of activation of the HA protein plays a key role in the pathogenicity and transmissibility of H5N1 influenza viruses in mallards. Natural H5N1 virus isolates are highly pathogenic in many, but not all, duck species (21, 47, 48), and their transmission among wild ducks and from wild ducks to domestic poultry and mammals, including humans, has been a key element in their natural ecology (10, 33, 54). Moreover, wild ducks are thought to be a main reservoir of low-pathogenicity avian influenza viruses (33). The intraspecies and interspecies transmission of influenza viruses depends on at least four factors: (i) the amount of virus shed by the donor, (ii) the stability of the virus in the environment over time, (iii) the time between donor shedding and acceptor exposure, and (iv) the infectivity of the virus in the acceptor animal. Since the pH of activation of the HA protein was found here to determine both the amount of shedding from ducks and the stability of virus in the environment, this molecular property may have an essential role in the propagation of H5N1 viruses in aquatic birds. Furthermore, HA mutations that maximize virus shedding and environmental stability via altered HA acid stability may be expected to promote both intraspecies and interspecies transmission. A broad survey of the environmental stability of 12 low-pathogenicity avian influenza viruses of various subtypes revealed that they were generally most stable at a slightly basic pH (7.4 to 8.2), a low temperature, and fresh to brackish salinity (1). The viruses lost infectivity much more rapidly after incubation under acidic conditions (pH <6.6), warmer temperatures, and higher salinity. Among the HA mutations characterized in the present study, the N1142K mutation increased the pH of activation to 6.4 while significantly reducing environmental stability, and the H241Q and K582I mutations reduced the pH of activation to 5.6 and 5.4, respectively, and moderately increased environmental stability. Thus, the pH of activation of the HA protein contributes to the duration of H5N1 influenza virus infectivity in the environment.
In the present study, an optimal range in the pH of activation of the HA protein supported the propagation of H5N1 influenza viruses in ducks. The adaptation of other subtypes of influenza viruses to different host tissues and species has been found to involve the selection of viruses with altered pH values for membrane fusion. A few passages of egg-grown recombinant X-31 influenza virus (H3N2 with the internal genes of A/PR/8/34 [H1N1]) in mammalian MDCK and Madin-Darby bovine kidney (MDBK) cells consistently resulted in HA protein mutations that increased the pH of HA-mediated membrane fusion from 5.2 to 5.6 to 5.8, and similar results were found after the passage of egg-grown A/Japan/305/57 (H2N2) virus in MDCK cells (25). The natural adaptation of H7N3 influenza viruses from wild ducks to turkeys coincided with two amino acid mutations in and near the HA2 stalk and a decrease in the pH of activation of the HA protein without a change in receptor binding (13). However, it is not known whether these mutations exclusively caused the reduction in the pH of membrane fusion, because the adaptation also resulted in a 23-amino-acid deletion in the NA stalk that reduced neuraminidase activity. We showed here that the absence of neuraminidase activity results in a lower pH of membrane fusion by the HA protein. Moreover, decreased neuraminidase activity in H3N2 influenza viruses has been shown to reduce virus entry (28, 32). In general, there may be a cooperative interaction between the neuraminidase activity of the NA protein and the fusogenicity of the HA protein. A functional balance between neuraminidase activity and the receptor binding activity of the HA protein is well known in many influenza virus subtypes (17, 20, 29, 30, 52).
In influenza viruses of the H3N2, H7N1, and H7N7 subtypes, an increase in the pH of activation of the HA protein results in resistance to high concentrations of amantadine (>0.1 mM), which raise the endosomal pH (6, 9, 18, 44). In a recombinant virus bearing the envelope glycoproteins of A/Netherlands/219/03 (H7N7), an HA-G232C mutation in the fusion peptide that reduced the pH of membrane fusion from 5.4 to 4.4 reduced in vitro replication by more than 2 log10 units and increased the 50% mouse lethal dose by more than 3 log10 units (18). Thus, in mammalian species there may also be an optimal range of HA protein activation pHs that supports efficient virus replication, infection, and pathogenicity. Since high- and low-pathogenicity influenza viruses differ in their tissue tropism in avian and mammalian species (24, 27), the optimum pH values at which their HA proteins are activated to support successful infection and transmission may differ according to the influenza virus and the host species. Future investigation of the biological properties of the recombinant viruses from the present study with mouse and ferret models may reveal whether changes in the pH of activation of the HA protein support the adaptation and transmission of H5N1 influenza viruses in mammalian species, which are significant factors in the pandemic potential of these viruses.
We thank Scott Krauss, Heather Forrest, David Carey, and Sharon Lokey for technical support in the BSL3+ facility. We thank Robert Lamb for the pCAGGS plasmid. We thank Rebecca DuBois and Stephen White for helpful discussions on protein structure and Sharon Naron for editing the manuscript. We thank the Hartwell Center for Bioinformatics & Biotechnology at St. Jude Children's Research Hospital for DNA sequencing.
This project was supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract HHSN266200700005C, and by the American Lebanese Syrian Associated Charities (ALSAC).
- Received 30 September 2009.
- Accepted 7 November 2009.
- Copyright © 2010 American Society for Microbiology


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OfficeHours Cards OfficeHoursCards is both a physical and digital product for helping people to prepare for their startup accelerator interview, or just to help refine any startups vision. It's a super simple business model with huge room for potential. Would suit someone who likes the startup world but doesn't want to have to manage an app or a team of developers. It featured on ProductHunt last October and got around 50 orders in 1 day. It continues to bring in orders each time there is any promotion of it. It has brought in thousands of dollars of profit so far and can easily be expanded upon. I am now consolidating all side projects to focus on new work. The sale includes :- Domain HTML & CSS site Design assets Supplier contacts Remaining inventory & branding materials worth $250 Future marketing suggestions documentation Contacts for graphic & web designers Assistance with transition and setup to new owner. It's pretty much a business in a box! Serious offers only please Built with: HTML, CSS, Gumroad $3000
Project description Dating social script, is the best way to start your own dating website! It is fast, secured, and it will be regularly updated Upload Images: Upload any image from your device and share it online with other users High Performance & Capability: PixelPhoto can handle any amount of vidoes / images ..This project is built usingPHPMySQLhtml5/javascripthtml5 & css3jQuerybootstrap 4 Detailed price: FOR SALE ⋅ > $800 (USD) Project type: Chat Online Dating Video Chat Posted by: other viewed 3 times posted by mbarkichadi Technology: This project is built usingPHPMySQLhtml5/javascripthtml5 & css3jQuerybootstrap 4
Browse by voice 'Browse by Voice' is an end-to-end speech based browsing application that captures user commands from your computer based audio input or your android device. Here is what you can do using 'Browse by Voice' - Check emails Send Chat messages Open websites Navigate websites Use search engines Play videos Open images Automatically login to your social media and email accounts All of these without using your keyboard or mouse. And this is not it. You can even connect your computer to your android device wirelessly and send voice commands via our android app. Built with: JavaScript, Chrome APIs $500
SendCatch.com Sendcatch.com is a neat little app that lets you transfer files between anything with a web browser and an internet connection. Go to https://sendcatch.com on two devices, and then connect any of the following ways: - Press 'Bump!' on two different devices, OR - Bump a mobile device with the spacebar, OR - Bump two devices together Here are all the ways you can connect: https://www.sendcatch.com/help DigitalOcean hosting is $5/month. Includes @sendcatch Twitter account. Built with: NodeJS, MongoDB $300
Project description Domain Registrar: Namesillo (easy to push) Reg Date: 2019-05-14.This project is built usingWordPress Detailed price: FOR SALE ⋅ > $200 (USD) Project type: Computers Hardware Wireless Posted by: website viewed 14 times posted by yasirgasim1 Technology: This project is built usingWordPress
Project description Get thorough answers for verifying your device from various infections, malware, and other online dangers at one single stage. Learn appropriate direction for downloading, introducing, and enacting the norton. com/setup. Detailed price: FOR SALE ⋅ > $99 (USD) Project type: Computers Posted by: website viewed 4 times posted by davisjohn64
Show HN: A device I built to improve my sleep
Show HN: Building an open-source and high-quality webcam with a Raspberry Pi 0 W
no description
Show HN: Raspberry Pi (or any Linux machine) monitor app
Show HN: Rewurk – A tool to help build websites that look great on any device
Show HN: Backboard Backpropagation: Physical Optimization Using JAX
Show HN: Chromium + Hardware accelerated Ray Tracing
Show HN: `downtimed` replacement for Raspberry Pi
Show HN: Palm Vein Authentication with a Raspberry Pi
no description
Show HN: YUV image stream from a V4L device (i.E. Webcam) using WebSockets
Show HN: Save notes from your desktop/mobile browser to Raspberry Pi
Show HN: Web controlled traffic light for Raspberry Pi powered by Elixir/Nerves
Show HN: Multi-room audio with Snapcast and Raspberry Pi

subset: commoncrawl


text: Mom arrived in New York from Holland at 2 years old with her parents and brother. She became a nurse. Like many immigrants, in WWII she volunteered to serve. In the Army Nurse Corps, she tended to wounded soldiers and the survivors of the Holocaust at Ebensee prison camp. She was awarded the Bronze Star for dedication to her patients when her field hospital was bombed. She's on the right in this photo – http://www.scrapbookpages.com/sachsenhausen/counterfeit.html


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3 Great Gravely OptionsApril 17, 2018
If you're familiar with Gravely, you already know comfort and precision are at the core of every product they create. They've been around for over 100 years, and with their obvious dedication to quality and innovation, it's hard to imagine they won't be around for 100 more.
Gravely offers products in three distinct categories:
- Stand-On Mowers – For those looking for quick maneuvering and intuitive operator features, Gravely's Pro-Stance mowers are stand-on mowers that feature an easy to adjust deck that ranges from 36 to 60 inches depending on your model. The flip-up suspension platform provides comfort to stand on, or folds up if you prefer to walk behind. A height adjustment system makes it more convenient for shorter mower operators. Gravely stand-on mowers have ground speeds of 8/3 mph for the 36" deck model, and 9/4 mph for all other models. These speedy machines will mow between 2.9 and 5.5 acres an hour. (Please note, we usually do not stock stand-on mowers but can order them.)
- Walk-Behind Equipment– Adaptable to any terrain, Gravely walk-behind equipment offers precision and durability to easily tackle the toughest jobs.
- Power Brush – Gravely's walk-behind Power Brush clears anything you throw its way. Dirt, grime, gravel, sand, snow, slush, salt and more will be cleared all the way to the ground with its pivoting brush and height adjustment system.
- Pro-QXT – If versatility is what you desire, it's hard to beat the Gravely Pro-QXT. The quick-attach system allows you to go from a blade, brush or snow blower to finish or brush mower easily.
- Pro-Walk Gear – These commercial lawn mowers require minimal maintenance and feature maximum performance. With a deck size of 32, 36, or 48" and a 5.4 mph ground speed, you can mow 1.7, 2, or 2.6 acres an hour. The 7-gauge deck, adjustable handlebars, and ergonomic controls make it easier on your crew.
- Pro-Walk Hydro – If you need a bit more power than the Pro-Walk Gear can offer, than the Pro-Walk Hydro may be more to your liking. This commercial mower features intuitive steering, a wide wheel base, and single-point height of cut that is adjustable in .25-inch increments. Deck sizes for the Pro-Walk Hydro range from 36-60" with a ground speed of 8/3 mph, and they can mow 4.3 acres an hour.
- Zero-Turn Mowers – The Gravely Zero-Turn lineup features ten different models and a range of accessories. Quickly go from mower to mulcher, or add a bagger, sunshade or other great accessories. The fully tubular frame design, designed deck leveling, and constant belt tension system are just a few features you won't want to miss. Deck sizes range from 34 to 72 inches.
If you're interested in learning more about any of these great Gravely product options, including Gravely Mowers, call us at 215-348-9041 or stop by Eagle Power Turf & Tractor, and we'll be happy to match you with the perfect machine. Promotional offers are available on Gravely's website.


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About Us
King Leopold Air first took to the Kimberley skies back in 1992 as a small charter company based on a pastoral station close to its namesake – the spectacular King Leopold Ranges. Today, the company benefits from an extensive aircraft engineering background and has seen much expansion in both the commercial and leisure tourism sectors. King Leopold Air's Head Office is located at Broome International Airport with operational bases at Derby and Cape Leveque.
Experienced pilots are additionally trained as tour guides in order to provide a comprehensive and informative insight into the stunning Kimberley landscape.
Our operations team and booking staff are hand picked for their local knowledge providing valuable assistance in itinerary and tour planning around the Kimberley and Pilbara.
All bookings and enquiries can be may be direct on our website, via email, telephone or by visiting our office at 9 Gus Winckel Rd, Broome International Airport. The office is open 9am till 5pm, 7 days a week.
Flights from Broome depart from the King Leopold Air Terminal.
We operate the largest fleet of GA8 Airvan, Cessna 206, 207, 210 and 182 aircraft in the West Kimberley, which are an ideal aircraft for the challenging Kimberley conditions. These are usually fitted with 5 passenger seats, the Airvan has 7, with a high wing design, greater stability and optimum viewing. Each passenger has a window seat and a personal headset to listen to the pilot's commentary. Pilots circle landmarks of interest, so that passengers maximise photo opportunities. These types of aircraft can also cover great distances very quickly, making them ideal for charters and tours into even the most remote and often inaccessible locations in the Kimberley and Pilbara.
King Leopold Air is your passport to The Kimberley, anywhere, we'll fly you there! With over 25 years of experience, we are committed to providing outstanding customer service and highly values its relationship with passengers. With a TripAdvisor Certificate of Excellence award five years running and recently awarded the 2019 Certificate of Excellence Hall of Fame Award, our aim is to exceed expectations while providing, through its operations
Flying to some of the most stunning locations in Australia, our passion is to connect people to the Kimberley. At King Leopold Air, we are very passionate about Aviation, Adventure Tourism and our collaboration with our Indigenous Tour Partners and The Kimberley and Pilbara region. Situated at the gateway to the Kimberley, Broome offers the perfect departure point for a Kimberley scenic adventure. Explore ancient fossilised reefs, and arid-land eco systems through spectacular gorges, ancient caves, rocky tunnels, crystal-clear waterways and cascading waterfalls.
From the World Heritage listed Bungle Bungles to ancient gorges of Karijini National Park or majestic Mitchell Falls, we've been flying the skies of Australia's remote northwest for more than 25 years. Fly the Horizontal Falls, Cape Leveque, Cygnet Bay Pearl Farm, Geikie Gorge, Bungle Bungles, Mitchell Falls, Windjana Gorge, Tunnel Creek, Mimbi Caves and Karijini National Park, the experiences are endless! Our scenic adventure flights are the best way to explore these amazing sights and experience the dramatic and beautiful landscapes of the Kimberley and Pilbara.
Every guest who flies with King Leopold Air will receive a souvenir 'Passport to the Kimberley' tour map, a souvenir tour photo to share with family and friends, a King Leopold Air stubby cooler and a complimentary Matso's beer voucher!


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Energy Efficiency Kit Program from
FirstEnergy's Ohio Utilities
The Energy Conservation Kit program formerly offered by your FirstEnergy Ohio utility has come to a close. For a list of the energy efficiency programs that are still available, please visit www.energysaveoh.com. If you have already received a kit and have questions about the components, please call 800-777-5655.


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LEASES; POSSESSION — Unless a residential lease says otherwise, the covenant of quite enjoyment means that the property has to be ready for the tenant to use and enjoy as its residence the same day that the lease provides the tenant may move into the property.
A residential lease provided that if there were any occupancy problems in a rental, single-family home, the landlord had fourteen days to address and correct those problems. After the lease was signed, the tenant went to the home with the intention of cleaning it before moving in, but there was no water. "Two days later, after the water had been turned on, she reentered the home and discovered a roach infestation." The tenant told the landlord about the problem and the landlord, though claiming to be unaware of the roaches, immediately arranged for exterminator services. A week later the landlord told her tenant that the house was ready, but after visiting the house, the tenant decided to terminate the lease because the infestation apparently had not been remedied. The landlord returned the security deposit but claimed the right to retain the first month's month because of the lease's provision giving her "fourteen days to address and correct any occupancy problem."
The tenant sued for return of the rent and the lower court ruled that "[w]hen a tenant signs the lease, the tenant is entitled to what we call qui[et] enjoyment. [This mean] [t]hat [the] property has to be ready for a tenant to use and enjoy as their residence the same day that the lease provides they move in." As a result, the lower court ordered that the rent be returned. On appeal, the landlord lost again. The Appellate Division rejected all procedural arguments and then looked at the lease. After doing so, it held that the lower court "was correct in [its] application of the law to the facts presented." According to the Court, the landlord "had no contractual basis on which to demand rent because [she] could not deliver lawful possession and quiet enjoyment of the premises." The Court agreed with the lower court that the lease's provision giving the landlord fourteen days to cure an occupancy problem only took effect once occupancy had been delivered.
Copyright ©2010. Meislik & Meislik. All rights reserved.


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And that's the second version of Pączki :)
It turned out that when no one was looking at me while working in the kitchen, doughnuts got twice as big as the previous ones ;P Oh, what can I do that I like xxl baking ;) Let it be cookies, bars of chocolate or mentioned doughnuts… They have to be big! :)
This time I tried this recipe out. It's a nice one, too.
Just do forgive me that this post is so twin to the yesterday's one. The thing is, the procedures while making doughnuts are so alike that I decided to be a bit lazy posting this time :)
I'm a teacher so grading time :)
4+ for the dough
5+ for the taste (outstanding!)
So now it's your choice ;)
Ingredients (gives about 30 big doughnuts):
- 1kg plain flour
- 21g instant yeast
- 120g sugar
- 1/2 tsp salt
- 100g butter
- 6 yolks
- 1 egg
- 500ml milk
- 40ml food spirit
- 1/2 vanilla bean (or 16g vanilla sugar)
- juice and zest from one lemon
- plum jam
- sunflower oil, for frying
Sieve the flour.
Add yeast and salt. Stir.
Heat up milk with sugar, but just a bit so that the sugar is more or less dissolved.
Melt the butter.
Cool a bit.
Whisk the yolks with an egg with a fork.
Add warm milk to the flour and start kneading.
Add the egg mass and knead, knead and knead more :)
Add food spirit, lemon juice and lemon zest… and knead more and more :)
Add melted butter at the end and knead the dough till it's pliable and non-sticky.
Place the dough in the bowl and leave it for doubling its size. It'll take about 1,5h.
Once it's doubled, knead it again shortly.
Stretch the dough using your hands or roll it. It can't be too thin, though.
Using a glass, cut round doughnuts from the dough.
Leave them for a moment on a sieved worktop so that they get a bit more fluffy.
Heat the oil to 175°C.
Fry the doughnuts on hot oil for several minutes each side.
Take them out and dry on a kitchen towel.
Using a kitchen filler, fill them with plum jam.
Still when they're warm, sprinkle with icing sugar.


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To manage in an environment where this complex of conditions exists is a challenge. As a PM, you are responsible and accountable to the client and sponsor for the successful outcome of your projects. That means getting them done on time, within budget and to the satisfaction of the stakeholders.
What can one do?
That is where management by accountability comes in. If there is a plan founded on a clear and practical approach, where the plan includes a set of activities with deliverables, dependencies, target dates and responsibility assignments, and there is a communications plan with regular progress reporting, then the PM has at least a chance at success. These are the requisites for management by accountability.
When people know that their task performance against commitments will be clearly reported, it is more likely that they will make commitments that they can meet and then meet them or clearly state why they haven't. Transparency promotes effective performance.
If reporting is regular and based on the activities in the project plan, that implies that tasks that are not completed by their due date will be identified and published as part of the project record. If tasks are associated with individual performers, then there is personal accountability.
In many organization cultures, this kind of reporting is resisted. Blaming Is confused with holding people accountable. Nobody wants to be the one who identifies the cause of project schedule slippage. Some see the reporting as too much overhead. Others passively resist by not doing the work of updating the status of their tasks.
We need to find a way to overcome cultural resistance and recognize the value of effective reporting. Reporting has to be simple and as automatic as possible. Project management tools make this possible. If the plan is well created and makes use of dynamic scheduling, reporting against the project baseline relatively simple. But there is still overhead. Someone has to enter actual completions for tasks, people have to explain their status, issues, risks and changes must be managed.
Getting effective reporting to be adopted in an organization is part of a culture change that comes when disciplined PM is implemented. The PM who is working in an immature environment and has little formal authority must convince the stakeholders at all levels that management by accountability is in the best interest of the organization and the individuals on the project who are committed to project success.
Don't forget to leave your comments below.


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What is the favour of the Lord? Some think it's when things always work according to their plan. I used to think that's what the favour of the Lord is. I've been awaken though, now I believe it is the will of the Lord for our lives perfectly working itself out for our good in God's way and His timings. This means the favour of the Lord can come in the form of rejection or heartbreaks. It's not always going to be all butterflies and smiles in the sunlight. Yea, sometimes it will hurt. How can it hurt and still be the favour of God? You'd ask. It's simple, we believe that God is all knowing and He certainly is. What we may think is best for our lives God knows we're way off course. So when we ask God for his favour, He does what He knows is best for us. What God knows is always more accurate than what we may think, imagine, calculate and all that stuff.
We're off to an interview, well prepared. We pray, God I thank you for your favour , which basically means we want to get through with the job, period. If we dont get the job we somehow doubt we have the favour of the Lord upon us.
Can I challenge us to change the way we think? Instead, when we pray for His favour, realise that whichever way it goes it is the favour of the Lord working in that situation. So if you don't get the job know that God is seeing the bigger picture He knows everything about the future and it happened that way for a reason.
Praying for God's favour upon your life is putting absolute trust in who God is. You let go of all your ideas and trust that what God does is the best for you. Trust God!
Easier said than done, it is. But can we "challenge" ourselves to really rely on God. Challenge being the operative word. The song writer Donnie McClurkin says it best:
II know that faith is easy when everything is going well
But can you still believe in Me when your life's a living hell?
And when all the things around you seem to quickly fade away
There's just one thing I really want to know
Will you let go? (I'll trust you, Lord)
Will you stand on My word? (I'll trust you, Lord)
Against all odds will you believe what I have said? (I'll trust You, Lord)
What seems impossible (I'll trust You, Lord)
Will you believe? (I'll trust You, Lord)
Every promise that I made will you receive? (Yes, I will trust You, Lord)
I know how bad it hurt you when that loved one's life came to an end
And when they had to leave you, you said you'd never love again
But will you trust that I can help you and I'll never turn away?
Will you trust Me, child, no matter, come what may?
What if it hurts? (I'll trust You, Lord)
What if you cry? (I'll trust You, Lord)
What if it doesn't work out the first time that you try? (I'll trust You, Lord)
What if you call My name (I'll trust You, lord)
And you don't feel Me near? (I'll trust You, Lord)
Will you believe in Me or will you fear? Oh, my child?
(Yes, I will trust, You Lord)
I will trust
I will trust
I will trust
Oh, oh, oh, oh know that faith is easy when everything is going well
Let us trust in God!
Prayer: Lord help me to trust you wholeheartedly when I pray for your favour.


text: A wet afternoon cried out for the warmth and dryness of a shelter made by Y7-9 during their recent Bushcraft experience. Not only did they provide shelter for themselves but cooked on an open fire and learned skills for life with regard to survival in the wild. Despite the weather they all had a great time and told the older students about their adventures well into the evening after tea.


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News & Activities
January Activities
January 14, 2019 /
Join us at City View for a New Year of activities. Sign up for Robbie Burns Day festivities. Friends of residents and the public are always welcome. Just call or drop by to register!
See below for this month's activities. PDF Download Here.


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Over the past few decades hemp has been making a big return. Consumers are recognizing the wonderful properties of hemp – for food, fuel, construction, textiles, plastics, and medicine.
In November of 1990, Hugh Downs talked about hemp, the history, the natural resources, and the political aspects. Below is the audio video.


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Paul Simon's song, "So Beautiful or So What," from his latest release is a great song for many reasons, and I will allow the song itself to mainly support my statement. I decided to post this song since today is the 45th anniversary of the assassination of Dr. Martin Luther King, and since the reference to that tragic event in this song is quite intriguing. I think the song's meaning (or purpose?) is summarized in the short question that is the chorus:
"So Beautiful, So beautiful, So what" Continue reading


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Tag Archives: Want2Scrap
Creating Dimensional Hippity Hop Easter Card
Happy Easter Greetings to you all! Margie Higuchi, here to share this sweet Easter card full of dimension using 3D Foam Squares and adhered with a combination of E-Z Runner® Grand and Adhesive Dots Micro to showcase an adorable dimensional … Continue reading
Many Birthday Blessings Card featuring Adhesive Sheets
Sending many birthday blessings through this layered card I created featuring Scrapbook Adhesives by 3L® Adhesives Sheets. Not only can you die cut with these sheets but you can easily carry them to your craft gatherings without the bulk of … Continue reading
Dimensional Mixed Media Background All Occasion Card Tutorial
Hello! Today I am sharing an all occasion card with a little bit of mixed media This is something easy to do – all you need is modeling paste. I will show you two different techniques for an easy and … Continue reading
With Love Tag and Jar and NSD Giveaway!
Happy Saturday! It's Judy here today to share an easy Mother's Day project. Actually, it could be used for other occasions as well. I've put together a fun tag to add to a mason jar filled with flowers. I'm using … Continue reading
Celebrate Shaped Card and Adhesive Sheets Tutorial
Hello~! With the new year approaching, I thought I'd share a sparkly and glittery new year card with you. I created a shaped card mainly put together with Adhesive Sheets – 6 x 12″ and touches of GOLD to welcome … Continue reading
3D Foam Shapes Holiday Pillow Box Tutorial
Greetings and Happy December! Today I'd like to share a gift wrapping idea using Scrapbook Adhesives by 3L® 3D Foam Shapes! Using a couple of techniques, I embellished the shapes to decorate this cute pillow box… Let me share how … Continue reading
Spellbinders Whimsical Woodland Blog Fest
Greetings! We are super excited to be participating in the Spellbinders® Whimsical Woodland Blog Fest and Partner Blog Hop today!!! Today we have 2 fabulous tutorials! Margie Higuchi and Laurel Seabrook are sharing some super cute stuff. Take a look, … Continue reading
Make Your 4th Sparkle with 3D Foam Stars
I am excited to share my FUN and simple card that I created with our 3D Foam Stars shapes. What a great way to celebrate America's birthday 🙂 Let's start with the card panel. I used three different patterned papers … Continue reading
Elegant and Simple 50th Anniversary Card Tutorial
It is wedding season which also means that anniversaries are coming up! I was asked to create a 50th wedding anniversary card – what an honor! I'd like to share how I created this beautiful 50th wedding anniversary card – … Continue reading


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In 1999, the surprising demand for the innovative living concept of the first Wohnfabrik in Toffen encouraged the designers to embark on their second project: the Wohnfabrik II in Steffisburg near Thun, a development almost four times larger than the original.
Close to the city centre of Thun, four apartment buildings include 61 apartments and a total floor space of 7740m2. Despite the size of the project, all apartments are designed according to the individual requirements of the owners.
During the millenium year of 2000, the first stage, the buildings Red and Blue, were completed and occupied. By 2002, the second stage, the buildings Yellow and Black, were finished. A housing estate was created that not only includes 61 individually designed apartments but a garage housing 60 cars as well as 9 covered and 11 visitor parking spots.
The grounds, the playground and the meeting places for young and old were designed using the ideas and suggestions of the residents, to ensure that the communal areas also reflected their individual characters.
Developer: Genossenschaft zur Wohnfabrik


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Due to the anniversary of Sharon Tate's death the Internet has been ripe with pictures seldom seen.
I love these. Sharon, timeless, ethereal...
Beautiful Sharon. She will always be timeless. I like to think she and Jay
would have ended up together. Jay, the man who loved her so, and died defending her.


text:
Gaïatrend expands into U.S.
Gaïatrend, a leading vapor company in France, is expanding into the United States, where it will be launching its flagship brand, Alfaliquid.Industry veteran and executive Arnaud Dumas de Rauly will lead Gaïatrend USA's development as president.
Currently commanding more than 50 percent of the e-liquid market in France, Alfaliquid's early success in Europe can be attributed to the brand's extensive variety of unique, quality e-liquid flavors and the company's commitment to health and safety regulations for each stage of production.
Gaïatrend will initially be rolling out 15 flavors in the U.S. market across Alfaliquid's three e-liquid collections, Heritage, Harvest and Reserve. Understanding the importance of transparency, particularly in an unregulated U.S. industry, Alfaliquid will also work to educate consumers about e-liquids and their ingredients as they have done successfully in France.
De Rauly is the former president of FIVAPE, a European vaping trade association. He currently serves as the organization's Secretary General for International Relations and is also a board member of the Vapor Technology Association, currently serving as Treasurer.
De Rauly also plays an integral role in the development of industry standards as a founding member of the AFNOR (French Standards Body) technical committee on vaping and vapor products, current member of the CEN (European Standards Body), and current member of the ISO International Standards Organization. He frequently serves as a keynote speaker at international vaping shows across the globe.
"We are thrilled to announce Gaïatrend's expansion to the U.S.," says De Rauly. "With the advent of a real vaping culture across the globe, we see tremendous opportunity for Alfaliquid in the U.S. Alfaliquid has set the industry standard in Europe through quality and innovation, and we look forward to introducing our e-liquid products to adult consumers in the states. With our expansion to the U.S., we will also work to educate adult consumers about e-liquids and their ingredients, as well as the importance of health and safety standards."
Alfaliquid e-liquids are manufactured by Gaïatrend in the French region of Lorraine.


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Imagine 2 sisters as close as close can be. Best friends who do everything together. Between us we have 5 children. We share everything, even having 2 of the 5 children diagnosed with 2 different forms of cancer only months apart from each other. This is our story:
In 2012, Angelas daughter, Alyssa, AKA Funky Monkey, was diagnosed with melanoma. We all watched as she was fighting for her life. Alyssa endured multiple surgeries to remove the cancer from her body. We soon discovered that her cancer was melanoma Stage 3A. Her Dr, Dr. Pappo, worked tirelessly to get Alyssa to NED (no evidence of disease). She is now a sophomore in High School and is thriving!
Being a native to Memphis, we grew up listening to stories of St. Jude from my grandmother & 2 aunts who were the First Ladies of St. Jude with Danny Thomas.We knew it was a great place, but had no idea we would need them to help us save our children. We walked into the doors of St. Jude with fear but also HOPE. We knew people came from all over the world to be treated at St. Jude.
We had no idea that our family would hear the words, "your child has cancer" on the morning of February 4, 2013...again.... It is a sentence no parent ever wants to hear and is makes your heat stop & that's the day time stopped for us. The words cause fear, anxiety, stress, & heartache. This time it was Michelles son, Tyler. Ty, who was only 6 at the time.
Ty began treatment the same day of his official diagnosis of ALL or Acute Lymphoblastic Leukemia. A 2-1/2 to 3 year treatment plan. He has endured chemo, blood, platelets, bone marrow, OT, PT, TONS of medications, rounds of testing, broken bones, school at St. Jude, the list goes on. St. Jude, Dr. Pui, A clinic staff, med room staff, chaplains, ALSAC, surgery room, recovery room, etc helped us walk this journey for 2 years and 7 months. We celebrated the end of treatment with a room full of friends and a NO MO CHEMO party. It was amazing. Even now, we go to St. Jude and are still seen for any side effect or worry we may have.
Because of St. Jude, we have our children. Our family stays complete. But, the fight is not over. We must fight until no child looses the battle. So every family that walks in the doors and hears the words "your child has cancer" has a fighting chance. St. Jude has never asked us for one thing over what our insurance didn't pay. They also made sure we had our focus on our child and not how we would pay our medical bills.
Over the past 7 years our team has grown and we have raised over $350,000 for the place that gave us something that you can NEVER put a price tag on, our children. Each year we have more Heroes join our team with the same mission in mind-To put an end to childhood cancer.
This year we will race again and raise money. Ty & Alyssa completed their second 10K last year for the place that saved their life. They say they did it for the kids who couldn't race and our 2 angels in heaven, Gage & Arianna. We will walk, crawl, run, skip, or hop side by side with St. Jude and ALSAC until no child dies in the dawn of life. We want to help complete the mission of Danny Thomas. Thank you for your love & support of St. Jude, ESP our TY & ALYSSA!


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Light and temperature provides the determining factor by which the body responds to the 24-hour activity period
It was believed that the fundamental locus or location of our biological clock is the brain specifically in the suprachiasmatic nucleus (SCN) of the hypothalamus.
In complex animals like humans, the hypothalamus forms part of the autonomous nervous system which in part, controls the functions of organs that are not voluntarily controlled, hence involuntary. Involuntary, meaning, they are not governed by conscious will to act. These include breathing, heart beat and intestinal digestion.
One interesting point to make is that the location of SCN is just above the optic chiasma (cross). The optic chiasma can be described as a location where optic nerve fibers meet and cross (chiasma-cross).
It is fitting to say that, the perception of light through the opening in the eyes, triggers the wake/sleep patterns in humans since this is one way the organism receives such physical message.
As the retina (a portion of the eye where initial image of an object is stored) traps light rays, optic fibers relays the image to the brain via characteristic electrical pulses.
This unique sensory impulse is carried along the optic fibers to the occipital lobe (rear part) of the brain and is perceived as images. The proximity of the optic chiasma to the SCN perhaps provides the opportunity for the hypothalamus to "sense" the referred electrical impulses.
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Internship: Office of Planning and Budget
The principal role in the Endowment for the Office of Planning and Budget (OPB) is the fiscal oversight and control of all agency appropriated funds. In this oversight, the office is responsible for the formulation, execution and annual review of these various appropriations and assurance that these funds are budgeted in accordance with both OMB and Congressional guidelines.
Interns would be responsible for assisting in either exercises related to the appropriate administration and execution of current appropriated funds or the budget exercises related to formulation of future budget year requests and reporting for OMB and Congress. In addition, interns would assist staff in budget research and writing exercises as appropriate to the individual's skill set or prior experience. Backgrounds in public administration or finance would be appropriate for these intern positions. In addition, demonstrated skillsets would include writing, math and knowledge of excel/budget spreadsheets.
If you have any questions, please contact the NEH Internship Coordinator at 202 606-8317 or @email. We look forward to hearing from you.


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Avis Sydney Airport Car and Truck Rental
Transport and Hire Mascot, Bayside, New South Wales
Avis is a leading car rental company in Australia, servicing both leisure and business travellers. Avis has been providing its customers with the famous 'We Try Harder' service for over 55 years and continually strives to improve the customer experience.
Avis has over 240 locations across Australia and has a wide range of the latest model vehicles to suit all your rental needs. When travelling in an unfamiliar city, don't forget to add Avis' portable GPS unit to your rental and never get lost again!
Qantas Frequent Flyers can earn points with Avis.
For more information please visit their website.
From compact cars to full-size sedans and luxury cars to convertibles, Avis Rent A Car has the rental vehicles to make your next trip a memorable one. To obtain more information on vehicles available, please visit their website.


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ASLTIP Speech and Language Therapist
Member Since
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My name is Mel and I specialise in working with preschool and primary aged children with Down Sydrome. I work in various areas along side a great team of therapists at 'Little Talkers LTd'.
If I am unable to help, I have a team I can pass you on to.
Copyright © 2003-2016 ASLTIP • All rights reserved
The Association of Speech and Language Therapists in Independent Practice - Company No. 07984835,
71-75 Shelton Street, Covent Garden, London, WC2H 9JQ, tel 0203 002 3704


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Growth capital private equity firm TA Associates has promoted Dhiraj Poddar as Principal and Director in the Mumbai office. Poddar joined the India office, TA Associates Advisory Pvt. Ltd, in 2010 from Standard Chartered Private Equity.
The promotion comes as TA Associates, which has invested in 425 companies globally and has raised $18 billion in capital, also announced promotions of Elizabeth de Saint-Aignan and Patrick Sader as Principals in its Boston and London offices, respectively.
"These team members have played an important role at our firm and reflect TA's talented team of professionals," said Richard D. Tadler, Chairman and a Managing Director at TA Associates. "Their strong experience investing in and guiding the growth of portfolio companies has created value for our Limited Partners, portfolio companies and TA."
Poddar has advised on TA's $40 million investment in Tega Industries Ltd, which designs and makes consumables for the mining industry and a minority stake in e-payment processing services provider BillDesk (part of IndiaIdeas.com). He also sits on the board of both companies.
Before Standard Chartered Private Equity, Poddar worked with Progeon, the ITES arm of Infosys, as part of the team that set up the KPO division. He has also served a stint with ICICI Securities as a assistant vice president. Poddar holds a degree from the Institute of Chartered Accountants of India and an MBA from the Indian Institute of Management, Ahmedabad.
(Edited by Prem Udayabhanu)


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Brookfield Mobile Home Park (GNIS FID: 1979572) is a populated place located within the Town of Brookfield, a minor civil division (MCD) of Worcester County.
The elevation of Brookfield Mobile Home Park is 728 feet. Brookfield Mobile Home Park appears on the Warren U.S. Geological Survey Map. Worcester County is in the Eastern Time Zone (UTC -5 hours).
Worcester County: Subdivisions (MCDs) | Cities & Towns | ZIP Codes | Features (airports, churches, hospitals, etc.)
Also See: Nearby Photos | Nearby Hotels | Driving Directions
BIG Map |
Driving Directions |
Terrain Map |
Satellite Map | Data Source - USGS GNIS FID: 1979572
BOUNDARY MAPS: Town of Brookfield | Worcester County
| Brookfield, MA (3.4 miles NNE)
Fiskdale, MA (3.9 miles SSE)
Warren, MA (4.3 miles NW)
West Brookfield, MA (4.5 miles N)
Sturbridge, MA (5.1 miles SSE)
| East Brookfield, MA (5.8 miles NE)
North Brookfield, MA (7.1 miles NNE)
Holland, MA (7.5 miles S)
Ware, MA (8.3 miles NW)
Spencer, MA (8.7 miles NE)
| Nonatomqua Mobile Home Park, MA (2.7 miles NE)
Little Rest, MA (3.6 miles SW)
East Brimfield, MA (4.3 miles SSW)
Marks Garrison, MA (4.7 miles NNW)
Old Sturbridge Village, MA (4.7 miles SSE)
| Brimfield, MA (4.9 miles SW)
Union Chapel, MA (5.1 miles E)
Robinson Crusoe Camp, MA (5.6 miles SSE)
West Warren, MA (6.1 miles WNW)
Mystic Grove, MA (6.6 miles E)