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Antibody responses to influenza virus hemagglutinin provide protection against infection and are well studied. Less is known about the human antibody responses to the second surface glycoprotein, neuraminidase. Here, we assessed human antibody reactivity to a panel of N1, N2, and influenza B virus neuraminidases in different age groups, including children, adults, and the elderly. Using enzyme-linked immunosorbent assays (ELISA), we determined the breadth, magnitude, and isotype distribution of neuraminidase antibody responses to historic, current, and avian strains, as well as to recent isolates to which these individuals have not been exposed. It appears that antibody levels against N1 neuraminidases were lower than those against N2 or B neuraminidases. The anti-neuraminidase antibody levels increased with age and were, in general, highest against strains that circulated during the childhood of the tested individuals, providing evidence for "original antigenic sin." Titers measured by ELISA correlated well with titers measured by the neuraminidase inhibition assays. However, in the case of the 2009 pandemic H1N1 virus, we found evidence of interference from antibodies binding to the conserved stalk domain of the hemagglutinin. In conclusion, we found that antibodies against the neuraminidase differ in magnitude and breadth between subtypes and age groups in the human population. (This study has been registered at ClinicalTrials.gov under registration no. NCT00336453, NCT00539981, and NCT00395174.)
IMPORTANCE Anti-neuraminidase antibodies can afford broad protection from influenza virus infection in animal models and humans. However, little is known about the breadth and magnitude of the anti-neuraminidase response in the human population. Here we assessed antibody levels of children, adults, and the elderly against a panel of N1, N2, and type B influenza virus neuraminidases. We demonstrated that antibody levels measured by ELISA correlate well with functional neuraminidase inhibition titers. This is an important finding since ELISA is a simpler method than functional assays and can be implemented in high-throughput settings to analyze large numbers of samples. Furthermore, we showed that low titers of broadly cross-reactive antibodies against neuraminidase are prevalent in humans. By the use of an appropriate vaccination strategy, these titers could potentially be boosted to levels that might provide broad protection from influenza virus infection.
Influenza virus infections remain a major public health concern worldwide. The available influenza virus vaccines represent valuable tools to prevent infection when the vaccine strains are well matched with the circulating strains (1). Immune responses induced by available inactivated influenza virus vaccines primarily target the immunodominant globular head domain of the hemagglutinin (HA) which is responsible for viral attachment and fusion. These antibodies (Abs) can be strongly neutralizing, but their target, the globular head domain, has inherently high plasticity (2). The virus has the ability to escape the immune response by introducing mutations into this domain, a phenomenon called antigenic drift.
The second major surface glycoprotein, the neuraminidase (NA), is often overlooked as a vaccine antigen (3, 4). The NA has enzymatic sialidase activity which is important for the transport of incoming virions through mucins and for the release of budding virus from infected cells (3). Inhibition of this sialidase activity is a major mechanism of action of NA-directed antibodies, but other mechanisms which involve antibody-effector cell interactions might be important as well. Inactivated influenza virus vaccines usually induce low immune responses to the NA (5–7), likely due to the immunosubdominance of the NA when it is presented to the immune system in conjunction with the HA (5, 8–10). In addition, NA tetramers are outnumbered on the influenza virus particles by HA spikes 1:4 (11). However, antibodies against the NA can protect from influenza virus infection in animal models (6, 12–23). In fact, anti-NA titers have been shown to confer protection from influenza virus infection and disease in humans as well (7, 8, 14). A recent human challenge study performed at the U.S. National Institutes of Health revealed that neuraminidase inhibition (NI) titers correlated well with protection against pandemic H1N1 disease symptoms and duration of shedding (24).
Here we describe the breadth, functionality, and subtypes/isotypes of anti-NA antibodies in children, adults, and the elderly. In addition, we explore the correlation between binding antibody titers measured in an enzyme-linked immunosorbent assays (ELISA) and levels of functional NI antibodies measured in an enzyme-linked lectin assay (ELLA) (25, 26).
Breadth, age dependence, and subtype/isotype of anti-NA titers.The breadth of anti-NA antibody titers in different age groups has not been extensively studied. Here, we used a panel of purified recombinant N1, N2, and influenza B virus NAs in a quantitative ELISA to measure antibody titers in children (6 to 59 months of age), adults (18 to 49 years of age), and the elderly (65 years of age and older). Sera were collected as part of clinical trials conducted with the influenza virus vaccine Flublok between 2006 and 2008, before the 2009 pandemic H1N1 strain began to circulate. NA titers (day 0) were visualized as heat maps and reactivity profiles (Fig. 1). Titers against N1 were generally low, with the strongest reactivity to the A/South Carolina/1/1918 (SC18, H1N1) N1 in the elderly and the A/USSR/92/1977 (USSR77, H1N1) N1 in the adults (Fig. 1A and D). The reactivity to a contemporary N1 from A/New Caledonia/20/1999 (NC99, H1N1) was lower, including in the youngest cohort. Furthermore, reactivity to the N1 of the pandemic A/California/04/2009 (Cal09, H1N1) virus, to which these individuals had not been exposed, was barely detectible. We also tested reactivity to the avian N1 of the A/Vietnam/1203/2004 (VN04, H5N1) strain and found low but detectible titers in the adults and elderly. Interestingly, the reactivity to the VN04 N1 was even slightly higher than against the Cal09 N1 for both groups. N2 titers were in general much higher than N1 titers (Fig. 1B and E). Again, the elderly had the strongest reactivity, with the highest titers against the pandemic A/Singapore/1/1957 (Sing57, H2N2) N2 and lower titers against more-modern isolates, suggesting a recall response to the earlier antigen, referred to as "antigenic sin" or back-boosting (27–29). Adults had a relatively high titer against the N2 from A/Philippines/2/1982 (Phil82, H3N2), with lower titers against older or more-recent isolates, suggesting that the first virus that they had encountered in their life had left an immunological imprint. Children had consistently low titers against all isolates. Interestingly, cross-reactivity to an N2 from a future (i.e., subsequently encountered) isolate, A/Hong Kong/4801/2014 (HK14, H3N2), was relatively high in both adults and the elderly and reactivity to an N2 from an avian isolate (A/chicken/Hong Kong/G9/1997 [ck97, H9N2]) was even higher, suggesting the presence of conserved epitopes. Titers against influenza B virus NAs were relatively high in adults and the elderly, with a distinct peak for the NA from the B/Yamagata/16/1988 (Yam88) virus and lower titers for B/Lee/1940 (Lee40) and more-recent isolates (Fig. 1C and F). Reactivity to a future isolate, B/Wisconsin/1/2010 (Wisc10), was relatively high, again suggesting the presence of conserved epitopes. Serum of children reacted poorly to the influenza B virus NAs.
We also examined the antibody subtype (see Fig. S1A to C in the supplemental material) and isotype (Fig. S1D to F) distribution of anti-N1, anti-N2, and anti-influenza B virus NA antibodies. In general, the pattern reflected the distribution seen with antibodies to HA (30–32), with the exception of elevated IgG4 titers against NC99 N1 and elevated IgG2 titers against Yam88 NB (Fig. S1). In summary, titers against N1 appeared to be lower than titers against N2 or influenza B virus NA; titers increased with age, and they were in general higher against strains that had circulated during the childhood/youth of the individuals in the adult and elderly cohorts.
Copyright © 2017 Rajendran et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license .
Correlation between binding and functionality of anti-NA antibodies.While binding of antibodies to the NA protein as assessed by ELISA is an indication of the presence of an immune response, it does not necessarily indicate that this immune response is functional and/or protective. Therefore, we measured functional antibody titers against four representative NAs using an enzyme-linked lectin assay (ELLA) to determine neuraminidase inhibition (NI) titers. These NAs included N1 from NC99, N2 from A/Panama/2007/1999 (Pan99, H3N2), influenza B virus NA from Yam88, and N1 NA from Cal09 virus (as an example of a future strain that these individuals had not been previously exposed to). To minimize anti-HA head antibody-based interference (25, 33), state-of-the-art NI assays using H6NX reassortants were employed. Humans have little or no H6 head-specific preexisting immunity; therefore, reassortant viruses that contain an H6 HA in combination with the NA of interest give a readout that is more reflective of the true NA-based NI titer (25).
As mentioned above, ELISA titers to NC99 N1 were low in children, highest in the adults, and slightly lower again in the elderly (Fig. 1A and Fig. 2A). NI titers measured in the ELLA using the H6N1NC99 virus reflected the results obtained by N1 ELISA. Children had the lowest titers, and titers of the adults and elderly were higher (Fig. 2B). No increase in ELISA or NI geometric mean titers from the pre-recombinant HA vaccination time point to the post-recombinant HA vaccination time point was detected (Fig. 2A and B), and we found good correlation between NI and ELISA titers, with a Spearman r value of 0.5549 (Fig. 2C).
ELISA titers for N2 NA from Pan99 were relatively low in children and higher in the older age groups. Here, we also measured ELISA titers of postvaccination sera and found no increase (as expected since the vaccine did not contain NA) (Fig. 2D). We then conducted an ELLA assay using an H6N2Pan99 virus. The measured titers followed the ELISA titers, with low levels for NI in children and higher levels for NI in the adults and elderly (Fig. 2E). Again, we performed an analysis and found that the ELISA titers against N2 significantly correlated with the H6N2Pan99 ELLA titers (r = 0.6981, Fig. 2F).
As described above and shown in Fig. 1, ELISA titers against the Yam88 influenza B virus NA were low in children but relatively high in the adults and the elderly (Fig. 2H). We also assessed the NI titers with an H6 reassortant virus. For the purpose of this study, we rescued a novel H6 virus that expresses the NA ectodomain of the B/Yamagata/16/88 virus (and the A/PR/8/34 NA noncoding regions, cytoplasmic tail domain, and transmembrane domain), which allowed us to measure NI titers without interference from anti-B HA antibodies (Fig. 2G). The NI titers determined with this virus were similar to the titers measured by ELISA, with no increases observed in sera from the prevaccination time point to the postvaccination time point (Fig. 2I). Again, we found good correlation between ELISA titers and H6NBYam88 NI titers (r = 0. 8643, Fig. 2J).
Prepandemic titers against the NA of the pandemic 2009 H1N1 virus are low.Subsequently, we assessed correlations between binding and NI for the N1 of the 2009 pandemic H1N1 virus. The serum samples were drawn from individuals in the three cohorts before 2009; thus, these individuals had therefore not yet been exposed to this virus. Not surprisingly, the ELISA titers measured with recombinant Cal09 N1 were very low in all age groups (Fig. 1A and Fig. 3A). Interestingly, the NI titers measured with H6N1Cal09 virus did not reflect the pattern seen with ELISA. While children and adults showed low titers as expected, the elderly cohort showed increased NI titers (Fig. 3B).
There was no significant correlation found between anti-NA ELISA titers and H6N1Cal09 NI titers (Fig. 3C). This suggests that the increased NI titers in the elderly were caused by interference from HA-specific antibodies. In an earlier study, we had investigated the prevalence of broadly protective anti-HA stalk antibodies in the same cohort and found higher anti-HA stalk titers in the elderly (30). These antibodies bind broadly to the conserved HA stalk and are also capable of binding to the H6 HA used in the H6NX viruses for the NIs. We therefore wondered if elevated titers of anti-HA stalk antibodies in the elderly might interfere with the NI assay in this cohort. We measured the reactivity of the sera to H6 HA and found an increase with age, with the elderly having the highest titers followed by the adults and with the children having the lowest titers (Fig. 3E). We correlated the anti-H6 titers with the H6N1Cal09 NI titers and indeed found a highly significant correlation (r = 0.726, Fig. 3F), indicating that anti-stalk/anti-H6 antibodies might contribute to the NI titers, specifically when low levels of NA-specific antibodies are present.
Anti-HA stalk antibodies interfere with NI assays.The correlation analysis provided indirect evidence for interference of anti-HA stalk antibodies in the ELLA-based NI assay. To further investigate this phenomenon, we performed ELLAs with wild-type (wt) and H6NX viruses in the presence of anti-HA stalk monoclonal antibodies (MAbs). All three tested anti-stalk MAbs showed NI activity to various degrees. Human anti-stalk MAb CR9114 binds to all known influenza A virus HAs as well as to influenza B virus HA (34). Used in the ELLA assay, the MAb inhibited the NA activity of all wild-type and H6NX viruses (Fig. 4A and D to I). Similarly, anti-stalk MAb KB2 (35, 36) had activity against all H1- and H6-expressing viruses (Fig. 4B and D and G to I). Finally, we also tested anti-H3-stalk MAb 12D1 (37), which showed low activity (Fig. 4C and E). While anti-NA antibodies inhibit neuraminidase activity by direct binding to NA (Fig. 4J), the NI activity of anti-HA stalk antibodies is most likely caused by steric hindrance (Fig. 4K) as previously shown for anti-stalk MAb 9H10 and H10N8 viruses (16). In summary, anti-HA stalk MAbs contribute to inhibition of NA activity in assays performed with wild-type and H6NX viruses, depending on the MAb used.
Immunity directed against the influenza virus NA has been shown to provide protection against viral infection in animal models (6, 12–23). Furthermore, several studies have suggested that NA-based immunity also correlates with protection from influenza virus infection and disease (7, 8, 14, 24). However, the breadth of anti-NA antibodies in the human population has not been extensively studied. Titers in adults and the elderly seemed to be highest against strains that these individuals had encountered early in life. As an example, the middle-aged cohort exhibited the highest N2 titer against Phil82 (H3N2), a virus that circulated when individuals in this cohort were younger than 15 years of age. Therefore, our data provide evidence for a phenomenon known as original antigenic sin, back-boosting, or antigenic imprinting which has been described in detail for influenza virus HA but not for the NA (27–29).
Interestingly, we found a striking difference between the titers against N1—which were relatively low—and the titers against N2 and influenza B virus NA, which were moderately high. This difference was detected with both the ELISA and the ELLA (NA inhibition), indicating that there might be an inherent difference in antigenicity between these NA subtypes. Reliance on this conclusion must be tempered by the fact that different substrates are used in the immunoassays involving N1 and N2. The higher titers for N2 may be a reflection of differences in stability between N1 and N2 NA. For the virus-based NI assay, the NA content of the virion might play a role as well, although a study by Tanimoto and colleagues did not find statistically significant differences in NA content among H1N1, H3N2, and influenza B viruses (38).
Adults and the elderly have high titers of anti-influenza B virus NA antibodies, while children are basically nonreactive, likely because they have not been exposed to influenza B virus. Increasing the influenza B virus NA titers in children by vaccination could improve protection against this infection (39).
When we examined the relationship between binding (ELISA) and functional (ELLA) antibodies, we found a good correlation for prepandemic seasonal N1, N2, and type influenza B virus NA. However, pH1N1 ELISA titers against N1, which were very low, did not correlate with NI titers. Here we present preliminary evidence suggesting that anti-HA stalk antibodies, which also bind to H6 HA of the reassortant viruses used in the ELLA, could be the cause of the low agreement between the two assays. So far, this phenomenon has been described only for H10N8 viruses and anti-stalk MAb 9H10 (16, 40). When three anti-HA stalk monoclonal antibodies were tested in NI assays with wild-type and H6NX viruses, all three showed activity to various degrees. This needs to be considered when NI activity is measured using the ELLA in sera from individuals who have HA stalk-specific antibodies.
In summary, we have assessed the breadth, functionality, and isotype/subtype usage of anti-NA antibodies in children, adults, and the elderly. Anti-NA titers increase with age, but the magnitude of the response is subtype dependent and appears to be low for N1 whereas it is higher and cross-reactive for N2 and influenza B virus NA. In addition, we provide evidence for original antigenic sin of the influenza virus neuraminidase based on the finding that the titers are always highest against the NAs of the viruses to which the subjects were first exposed. It will be interesting to determine in detail in future studies how anti-NA titers are shaped by natural infection and vaccination and how adjuvants would influence vaccine-induced anti-NA immunity. Understanding the characteristics of the NA-specific response will help researchers to design vaccines that induce high and cross-reactive anti-NA antibody titers in all age groups and against all subtypes.
Cells and viruses.Madin-Darby canine kidney (MDCK) cells were grown in complete Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Inc.). DMEM was supplemented with 10% fetal bovine serum (FBS; HyClone, Sigma-Aldrich) and a mixture of antibiotics (penicillin [100 U/ml] and streptomycin [100 µg/ml] [Pen-Strep]; Gibco). Sf9 insect cells were grown in TNM-FH insect cell medium (Gemini Bioproducts) supplemented with 10% FBS and Pen-Strep antibiotics. BTI-TN-5B1-4 (High Five) cells (Vienna Institute of Biotechnology subclone [41]) were grown in serum-free SFX medium (HyClone) supplemented with Pen-Strep. Viruses used in this study include wild-type (wt) strains pH1N1 (A/Netherlands/04/2009 [NL09]—antigenically equivalent to Cal09), H1N1 (A/New Caledonia/20/1999 [NC99]), H3N2 (HA and NA from A/Panama/2007/1999 [Pan99] and internal genes from A/Puerto Rico/8/34 [PR8, H1N1]), and B (B/Yamagata/16/1988 [Yam88]) and reassortant strains H6N1Cal09 (HA from A/turkey/Massachusetts/3740/1965 [Mass65, H6N2], NA from A/California/04/2009 [Cal09, pH1N1], and internal genes from PR8), H6N1NC99 (HA from Mass65, NA from NC99, and internal genes from PR8), H6N2Pan99 (HA from Mass65, NA from Pan99, and internal genes from PR8), H6N2Uru07 (HA from Mass65, NA from A/Uruguay/716/2007, and internal genes from PR8), H6N2Tx12 (HA from Mass65, NA from A/Texas/50/2012, and internal genes from PR8), and H6NBYam88 (HA from A/mallard/Sweden/82/2002 [H6N1], NA from B Yam88, and internal genes from PR8).
The novel H6NBYam88 virus was rescued through reverse genetics by use of an eight-plasmid system. The NA segment was generated by using In-Fusion cloning (Clontech). The packaging signals (noncoding regions, cytoplasmic tail domain, and transmembrane domain) for this clone were derived from PR8 N1, while the ectodomain used was from Yam88 NB. The HA used was derived from A/mallard/Sweden/82/2002, and the other six segments (PB2, PB1, PA, NP, M, and NS) were derived from PR8. All segments were cloned into a pDZ vector, a bidirectional transcription plasmid that utilizes polymerase (Pol) I and Pol II promoters. 293T cells were transfected with 500 ng of plasmids for each influenza virus segment using Lipofectamine 2000 (Thermo Fisher Scientific). After 24 h, cells and supernatant were collected and injected into 8-day-old embryonated chicken eggs. Viral rescue cultures were initially screened by performing a hemagglutination assay. The resulting virus was plaque purified and expanded in embryonated chicken eggs.
The viruses described above were grown in 8-to-10-day-old embryonated chicken eggs (Charles River Laboratories, Inc.). Inoculated eggs with wild-type influenza A and reassortant viruses were incubated at 37°C for 48 h, and wt influenza B viruses were incubated at 33°C for 72 h. Eggs were then cooled to 4°C for 4 to 12 h, harvested, and clarified by low-speed centrifugation (3,000 rpm, 20 min). Virus titers were determined by plaque assay on MDCK cells in the presence of tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin.
Recombinant HA and NA proteins.All recombinant proteins were expressed in BTI-TN-5B1-4 cells and purified from cell culture supernatant as previously described (6, 42, 43). Recombinant NA and HA proteins used in this study were derived from the following isolates: A/South Carolina/1/1918 (N1), A/USSR/90/1977 (N1), A/New Caledonia/20/1999 (N1), A/Vietnam/1203/2004 (N1), A/California/04/2009 (N1), A/Singapore/1/1957 (N2), A/Hong Kong/1/1968 (N2), A/Philippines/2/1982 (N2), A/chicken/Hong Kong/G9/1997 (N2), A/Panama/2007/1999 (N2), A/Hong Kong/4801/2014 (N2), B/Lee/1940 B (NA), B/Yamagata/16/1988 (NB), B/Malaysia/2506/2004 (Mal04, NB), B/Florida/4/2006 (Flor06, NB), B/Wisconsin/01/2010 (Wisc10, NB), and A/mallard/Sweden/82/2002 (H6). Briefly, recombinant baculoviruses expressing soluble proteins were propagated in Sf9 insect cells. The recombinant baculoviruses were used to infect BTI-TN-5B1-4 cells at a multiplicity of infection of 10 to express secreted soluble recombinant proteins that were then purified from cell culture supernatants using nickel-nitrilotriacetic acid (Ni-NTA) resin (Qiagen) as described previously (42).
Human serum samples.Human serum samples were obtained from clinical trials performed by the Protein Sciences Corporation. The trials were randomized, double-blind, multicentered trials determining the safety, reactogenicity, and immunogenicity of Flublok, a recombinant influenza virus vaccine. All participants in the trial received 135 µg of recombinant HA (45 µg of A/New Caledonia/20/1999-like H1, 45 µg of A/Wisconsin/67/2005-like H3, and 45 µg of B/Ohio/01/2005-like B HA). The trials were performed in three different cohorts: children (6 to 59 months of age), adults (18 to 49 years of age), and elderly (≥65 years of age). Human serum sample collection was completed between May 2006 and May 2008. Further information regarding the clinical trials can be found on ClinicalTrials.gov with the following identifiers: NCT00336453 (children [44]), NCT00539981 (adults [45]), and NCT00395174 (elderly [46]). Serum samples were obtained prevaccination (day 0) and postvaccination (day 28) based on sample availability for both time points and were then subsequently analyzed at the Icahn School of Medicine at Mount Sinai, New York, NY (protocol/exempt determination number 15-00126). Commercially available postpandemic 2009 serum samples (Innovative Research), collected in 2014, with no identifying information other than age, sex, and race were also used in this study.
Enzyme-linked immunosorbent assay (ELISA).Microtiter 96-well plates (Immulon 4 HBX; Thermo Fisher Scientific) were coated with 2 µg/ml HA or NA protein (50 µl/well) diluted in coating solution (KPL) and were then incubated overnight at 4°C. The following day, plates were washed three times with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS-T) and then blocked with PBS-T containing 3% goat serum (Life Technologies, Inc.) and 0.5% milk powder (blocking solution). Blocking solution was discarded after 1 h of incubation at 20°C. Serum samples diluted to a starting concentration of 1:100 were first added to the plates and then serially diluted 1:2 in blocking solution. The final volume in all wells after dilution was 100 µl. After a 2-h incubation period at 20°C, plates were washed 3 times with PBS-T. Secondary antibody (50 µl/well) diluted in blocking solution was added, and the plates were incubated for 1 h at 20°C and washed four times with PBS-T. After developing for 10 min with SigmaFast o-phenylenediamine dihydrochloride (OPD; Sigma), the reaction was stopped with 3 M hydrochloric acid. The plates were immediately read at an optical density (OD) of 490 nm using a Synergy H1 hybrid multimode microplate reader (BioTek). The background of each plate (and 3 standard deviations) was calculated for each plate and used as the lower limit for area-under-the-curve analysis using Prism 7 (GraphPad). The following secondary antibodies were used: anti-human IgG (Fab-specific) horseradish peroxidase (HRP) antibody (Sigma A0293) (1:3,000), anti-human IgA (α-chain-specific) HRP antibody (Sigma A0295) (1:3,000), anti-human IgM (μ-chain-specific) HRP antibody (Sigma A6907) (1:3,000), anti-human IgG1 Fc-HRP (Southern Biotech 9054-05) (1:3,000), anti-human IgG2 Fc-HRP (Southern Biotech 9060-05) (1:3,000), anti-human IgG3hinge-HRP (Southern Biotech 9210-05) (1:3,000), and anti-human IgG4 Fc-HRP (Southern Biotech 9200-05) (1:10,000). All serum samples were tested using anti-human IgG (Fab-specific) HRP antibody. To find differences in antibody subtype/isotype specificity (IgG1, IgG2, IgG3, IgG4, IgA, IgM), 20 individuals of each age group (day 0; prevaccination) were randomly selected and tested for all IgG subtypes, IgM, and IgA using NC99 N1, Pan99 N2, and B Yam88 NA substrates.
Receptor-destroying enzyme (RDE) treatment.Human serum samples were treated with receptor-destroying enzyme (RDE) (Denka Seiken) to remove nonspecific/background NA inhibition. A 25-μl volume of human serum was added to 75 µl of RDE. The mixture was incubated at 37°C for 16 to 18 h. RDE was inactivated with 75 µl of 2.5% sodium citrate and then heat inactivated at 57°C for 30 min. Finally, 75 µl of PBS was added to give a final human serum sample concentration of 1:10.
Enzyme-linked lectin assay (ELLA) to determine neuraminidase inhibition (NI).The ELLA assay was performed according to previously published protocols (25, 26). First, a neuraminidase assay (NA) was performed to determine the optimal virus concentration to be used in the NI assay. The NA assay was performed for all virus stocks. Microtiter 96-well plates (Immulon 4 HBX; Thermo Fisher Scientific) were coated with 25 µg/ml (100 µl/well) of fetuin (Sigma) diluted in coating solution (KPL) and incubated overnight at 4°C. The following day, virus stocks were serially diluted 1:2 in PBS containing 5% bovine serum albumin (blocking solution) in a separate 96-well plate, starting with the undiluted stock. The final volume in all wells after dilution was 150 µl. The plates were incubated at room temperature for 1.5 h. During the incubation time, the fetuin-coated plates from the previous day were washed three times with PBS containing 0.1% Tween 20 (PBS-T) and then blocked with blocking solution for 1 h at room temperature. After blocking was performed, the fetuin-coated plates were again washed three times with PBS-T. Viral dilutions (100 µl) were then transferred to the fetuin-coated plates and incubated at 37°C for 2 h. The plates were washed four times with PBS-T. Peanut agglutinin conjugated to HRP (PNA-HRP; Sigma) at a concentration of 5 µg/ml diluted in PBS (100 µl/well) was added, and the reaction mixture was incubated in the dark at room temperate for 1.5 h. After incubation, the plates were washed again four times with PBS-T and developed for 5 min with SigmaFast OPD (Sigma). The developing process was stopped with 3 M hydrochloric acid, and the reaction was immediately read at an optical density (OD) of 490 nm using a Synergy H1 hybrid multimode microplate reader (BioTek). To determine the optimal virus concentration to be used in the NI assay, the absorbance data were plotted in GraphPad Prism 7. The data were fitted to a nonlinear regression curve to determine the 50% effective concentration (EC50) and the 90% effective concentration (EC90). The EC90 was subsequently used for NI assays.
For NI assays, microtiter 96-well plates (Immulon 4 HBX; Thermo Fisher Scientific) were coated using a method similar to that employed for the NA assay. The next day, RDE-treated human serum samples were serially diluted 1:2 in separate 96-well plates, with a starting concentration of 1:10. The final volume of diluted serum samples in all wells was 75 µl. Virus stocks were diluted in PBS to the determined EC90 concentration. After virus (75 µl/well) was added to the serum plates, the plates were incubated at room temperature for 1.5 h. The fetuin-coated plates were blocked and then washed under conditions similar to those used for the NA assay. A 100-µl volume of the virus/serum mixture was transferred to the washed fetuin-coated plates, which were then incubated for 2 h at 37°C. The plates were washed four times with PBS-T, and PNA-HRP (Sigma) diluted to 5 µg/ml in PBS (100 µl/well) was added. The NA assay protocol was followed for the remaining NI assay steps. The human serum sample reactivity was determined by subtracting the background absorbance value from the raw absorbance value of human serum samples. The obtained value was divided by the average value from virus-only control wells and then multiplied by a factor of 100 to calculate the NA activity. Percent NI was determined by subtracting NA activity from 100%. Using GraphPad Prism 7, the percent NI was fitted to a nonlinear regression to determine the 50% inhibitory concentration (IC50) of the serum samples.
Statistical analysis.GraphPad Prism 7 was used to perform all statistical analyses. The three different age groups were compared using one-way analysis of variance with Tukey multiple-comparison tests. A Spearman correlation test was used for all correlation analyses. ELISA and ELLA data are shown as geometric means. Statistical significance is indicated as follows: n.s. (not significant), P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. The phylogenetic trees used in this study were built using ClustalOmega, and data were visualized using FigTree. The amino sequences for the NA proteins were obtained using GenBank and the Global Initiative on Sharing All Influenza Data (GISAID).
We thank Ariana Hirsh for technical assistance with protein expression and purification and St. Jude Children's Hospital for providing reverse genetics plasmids to generate H6N1 and H6N2 reassortant viruses.
This project was partially supported by the NIH Centers of Excellence in Influenza Virus Research and Surveillance (CEIRS) (contract number HHSN272201400008C to P.P. and F.K.), U19 AI109946-01 (to P.P. and F.K.), and R01 AI117287-01A1 (to F.K.).
- Received 16 December 2016
- Accepted 23 February 2017
- Published 21 March 2017
- Copyright © 2017 Rajendran et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license .


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When Christina first came to Shepherd's Door, she was paranoid. From the day she stepped into the facility, she was afraid of everyone, and she found every day to be absolutely terrifying as a result.
"Before I came here, I was caught up in a life of addiction and alcoholism," she said. "There were so many times when I would always make bad choices and get myself in unhealthy relationships. I was out of my mind because of my use of meth."
Since then, Christina has not only been 16 months clean and sober, but her life looks nothing like it did when she started in New Life Ministries.
"It's only by the grace of God that I'm still alive and here with my family," she emphasized in her speech to the crowd at Jubilee. "I don't react to my children the way I used to, I can actually talk without cussing, I can look my mom and Brian [stepdad] in the eyes today, and it's all because of God. My life has changed tremendously."
These dramatic changes in Christina's life were illuminated when she decided she needed to give back, and go serve downtown on a monthly basis with a group of ladies from Shepherd's Door at the Burnside Shelter. It was there she got a chance to really press in to her recovery, and tap even deeper into Jesus Christ – which she says has been an anchor for her soul since she was 13 years old.
"Never did I think that getting so far after being so low would bring me to the place where I could serve other people," Christina said. "I was able to go there, give my testimony in front of a bunch of people I didn't know, and it has made me so happy.
"I am now not only really comfortable in front of people, but I'm so thankful for these opportunities to serve."
And to the great benefit of Shepherd's Door, it has helped solidify her decision to stay on for a second year in our vocational training program we call Service in our New Life Ministries at Portland Rescue Mission.
TRAVIS AND TERESA VICE ON CHRISTINA'S RECOVERY JOURNEY OVER THE LAST YEAR (Travis is a pastor at Roots Community; Teresa is his mom):
I've been so encouraged about what I see in your life Christina. You changed before our very eyes. Not overnight like a wildflower popping out of nowhere, but over months like a tree slowly reaching to the sky, while seeking roots deep into the soil. Your identity has been transformed from a woman fearful and distant to one confident and vulnerable. Confident that Jesus has, and is, reshaping your story to be about His redemption and His mercy, not about your mistakes and brokenness. We're so proud of you Christina, and are with you in your journey, step by step with Christ.
Isaiah 40:31 (NLT) – "But those who trust in the Lord will find new strength. They will soar high on wings like eagles. They will run and not grow weary. They will walk and not faint."
- To help more women like Christina in need, consider giving a special gift today.
- Know someone in life crisis? Help them enroll at Shepherd's Door.
- Read about all seven of our most recent New Life Ministries graduates.
- "Rachelle escapes past, becomes 'new creation' at Shepherd's Door"
- "Elizabeth overcomes paralyzing grief, hopelesness at Shepherd's Door
- "Brianne discovers her true identity at Shepherd's Door"
- "Consumed by vengeance no more, Julie finds family at Shepherd's Door"
- "Tia overcomes destructive past, becomes 'miracle' at Shepherd's Door"
- "Tiffany unearths the desires of her heart at Shepherd's Door"


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'This is unconstitutional': Outrage over NEET highlights poor implementation of OBC reservations
A political storm has broken out over allegations that the quota of seats in medical and dental colleges available to members of the Other Backward Classes under the All India Quota has been drastically reduced.
Under the provisions of the National Eligibility, cum Entrance Test – which candidates seeking admission to medical or dental colleges anywhere in India must take – state government and private medical and dental colleges must surrender 15% of the seats in diploma and undergraduate courses for an All India Quota. Candidates from across the country can apply for this quota. In postgraduate courses, private and state government colleges must surrender 50% of the seats for the quota.
The controversy began to snowball on May 11, when the All India Federation of Other Backward Classes Employees' Welfare Association wrote to the National Commission of Backward Classes claiming that since 2017, candidates from the Other Backward Classes have been denied reservations under the All India Quota both in undergraduate and postgraduate seats.
On Tuesday, the Tamil Nadu unit of the Congress filed a Public Interest Litigation in the Supreme Court seeking a stay on admissions to undergraduate medical courses in the state since OBCs were being denied 50% reservation in the seats...
from Scroll.in https://scroll.in https://scroll.in/article/963802/this-is-unconstitutional-outrage-over-neet-highlights-poor-implementation-of-obc-reservations?utm_source=rss&utm_medium=public
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text:
Soha Ali Khan learns martial arts to essay a police inspector
Soha Ali Khan is playing the role of a police inspector in a film. To essay her character to perfection, she is learning martial arts from B-Town trainer Chitah Yajness Shetty
"Soha is very dedicated and hardworking. Every girl should learn the art for self defence so that they can defend themselves, as the police appears to be having a problem reaching everywhere," says Shetty.
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text:
Anders Breivik: Prison Treatment Is 'Inhuman'
Anders Behring Breivik, the far-right extremist who murdered 77 people in Norway in 2011, has said his treatment in prison is "inhuman."
Breivik, who is held in a high-security jail in Norway and has his contact with other people inside and outside prison strictly limited, is suing the Norwegian state over his treatment, which he claims is in contravention of the European Convention on Human Rights.
Norway's Attorney General has dismissed Breivik's complaints and strongly defended his treatment. A document submitted to Oslo District court reportedly said that the killer has access to three cells to allow him space to study, sleep and exercise.
"There are limits to his contact with the outside world which are of course strict — it pretty much has to be that way — but he is not totally excluded from all contact with other people," Marius Emberland, the lawyer who will defend the state at the hearing, told AFP. Breivik interacts with guards and professional staff but not other inmates, Sky News reported.
Breivik's lawyer said he suffers from "clear isolation damage." He is also claiming the state is not respecting his right to a private and family life.
A hearing will be held at the prison later this month. Breivik was sentenced to 21 years in jail in August 2012.


text:
Join us on this Escorted Private Tour of Carmel and Monterey and discover the magical rugged California Coastline, Scenic Views of Cliffs and Sandy Beaches and you will drive thought the unparalleled 17-Mile Drive!
Private tour departing from your hotel in San Francisco to visit the Napa Valley, famous wine region with a visit to some of the most prestigious wineries of the approximately 300 present in the valleys. Enjoy the view of the hills covered with vineyards and you will visit the historic district of Sonoma, where even today there is a testimony of the Spanish occupation: the Franciscan Mission.
Half Day Private Escorted Visit to Muir and Sausalito is ideal for all visitors looking to visit in more depth Marin County and the incredible Muir Woods National Monument with the tallest Redwoods! Visit also the charming Mediterranean style village of Sausalito famous for its wealthy population.
Half Day Private Escorted City Tour of San Francisco is ideal for all first-time and returning Visitors that would like to visit the city at their own pace! One of our official Tour Guide will pick you up directly at your Hotel to start the excursion.


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Herts SG14 2QH | 01992 551678 or 07929 950701
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Yes, we have heard you!
What is the point of having the best loose tea at home when you have to drink these horrible tea bags at work.
And here, ladies and gentlemen, is the answer to your problem:
Make your own personal tea bag with your favourite loose tea!
Simply fill the bag with the loose tea of your choice, then carefully pull the string and close it. Job done.
You now have a"normal" tea bag which you can take to work with you. Or camping. Or on holidays. Or… or… or… the possibilities are endless.
Use these fantastic DIY teabags to make some of them in advance and store them in one of our lovely tea caddies or any other storage box which you can leave in the office drawer or where ever you will need to store it.
Each pack contains 64 tea bags. .


text:
First UK property sold via Instagram
Given the popularity of the platform within the lifestyle sector it seems astonishing it's taken this long, but that's the story. A £320k one-bedroom property in a Kew Bridge development, marketed via Instagram by developers Apt Living, is the first UK property to be sold via Instagram. Not bad for an account with just 31 posts and 253 followers. More details here. And if you want to know what the Apt Living Instafeed looks like go here.


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Chapter 301 Plan No.1
Garnett was not only the second largest shareholder of Wilson's Company, but also the heir to the Garnett family in M Country. Last time, after Davis had failed to execute the killer plan, he had reported to Garnett about the Blood Revival Oral Liquid. They had discussed how to deal with Qin Haodong.
He touched his beard, looking a little confused. Then, he said, "Kim Lunhai's mask cost us millions. It's made of the latest nanomaterial and was produced with the most advanced 3D printing technology. It should've been flawless.
Even the facial infrared recognition system and the experts who made the mask failed to find the issue. How did the Celestial Empire People figure it out?"
Davis said, "Is it possible that Kim Lunhai has appeared somewhere?"
Garnett shook his head and said, "That makes no sense. Kim Lunhai has been doing this for decades. He is the most famous commercial spy in the field. It's rare for him to be found that soon."
Davis said, "I told you that the Celestial Empire guy was much sneakier than he looked. That's why I suggested that you get rid of him with Plan No.2."
Garnett said, "Plan No.2 would take too long, and we can't afford to wait. We'll suffer massive loss once the Blood Revival Elixir enters the market. We'll have to bring down Tangmen Traditional Chinese Medicine Company as soon as possible. Therefore, I insist that we should apply Plan No.1."
Davis shrugged and said, "But, dude, Plan No.1 has failed."
Garnett said, "No… no… I didn't use our perfect Plan No.1 because I didn't want our strength exposed. We'll definitely make it as long as I play my trump card."
Davis argued, "But we've already started preparing for Plan No. 2."
Garnett patted him on the shoulder and said, "Don't worry. Trust me, I'll definitely make it work this time. We'll switch to Plan No. 2 if I fail!"
Davis said, "Fine by me. I'll wait for two more days, but I don't think Plan No.1 will work. That Celestial Empire guy is too sneaky."
After the press conference of Tangmen Traditional Chinese Medicine Company, the whole pharmaceutical industry was in full swing. The success of the Blood Revival Elixir stirred the pond.
The on-site treatment of hundreds of patients at the press conference had caused the best publicity effect possible: No one doubted the effect of the drug anymore.
The next day, ten retail stores in Jiangnan City started to sell the Blood Revival Elixir. Due to the limited production capacity, the supply was restricted. There was a long line waiting in front of each store.
When Qin Haodong got back to work, he first inspected the production workshop and made sure the production line was working normally. Then, he turned back and walked to the office building after seeing everything go well.
The lobby of the office building of Tangmen Traditional Chinese Medicine Company was currently occupied by a crowd that spoke different dialects of the Celestial Empire. Some people were even foreigners with different skin colors.
They came from different pharmaceutical companies all over the world. After the Blood Revival Elixir had entered the market the previous day, those companies, who had a high business sense, had acted without delay. At once, they had come to Tangmen Traditional Chinese Medicine Company for the dealership of the drug.
Those people had been in the industry for many years and had seen the value of the Blood Revival Elixir immediately. They would get a great deal with large profits if they could get the dealership of the drug.
Two more clerks had been employed for reception work at the front desk, but they were still too busy.
"Hey, gorgeous. Please take a message for President Lin. Tell him everything will be negotiable as long as we get the dealership."
A middle-aged medical representative wearing glasses spoke.
The clerk explained patiently, "Sorry, sir. President Lin said that our medicine is now in short supply, so we don't need any representatives."
She was telling the truth. The products of Tangmen Traditional Chinese Medicine Company were different from other companies' products. They had set up ten retail stores, but the customers were still unsatisfied. There was no way that they would need someone to represent them.
"Please, gorgeous. Please tell President Lin I only need ten minutes with her. No… How about five minutes?"
The guy wouldn't stop trying, as he thought he could earn this opportunity with his eloquence as long as he could meet Lin Momo. Therefore, he thrust an envelope filled with money into the clerk's hand.
The clerk had not seen this coming. She blushed and gave the red envelope back. Then, she said, "I can't take this, sir. President Lin is too busy today. Her schedule is full already!"
Upon seeing that there was no hope, the guy could only take the envelope and leave in embarrassment.
When he left, another young man in his thirties came in. He was not like other people. He was not humble or cooperative at all. On the contrary, his head was raised high, and he looked quite arrogant.
The man glanced at the clerk at the front desk and said bossily, "I'm Zhao Kai, the head of Mancherk Company's Celestial Empire Branch. Tell President Lin that I'd like to see her now."
The clerk smiled professionally and asked, "Mr. Zhao, do you have an appointment?"
Zhao Kai's expression changed suddenly. He exclaimed as if he had been insulted greatly, "Have you been trained or not? Did you hear what I said? I'm the head of Mancherk Company's Celestial Empire Branch. Do I need an appointment to see President Lin?"
Although the clerk was scared of his attitude, she insisted, "Sorry, sir. According to the regulations, you have to make an appointment in advance, or you won't be able to see her!"
"What did you say?" Zhao Kaiqi slapped the desk and yelled, "I'm the head of Mancherk Company's Celestial Empire branch and I'm here for the dealership. You won't possibly be able to bear the consequences if this collaboration is delayed."
"Well… "
The clerk's face froze. She would totally have turned the guy down if he had been from another company. After all, rules were rules. However, Mancherk Company was a pharmaceutical giant who held 60% of the global drug chain. It was possible that she would be punished for pissing Zhao Kai off.
Upon seeing her get scared, Zhao Kai added, "Call President Lin now and ask her if she wants to see me. I'll leave if she doesn't."
"Fine then. Please hold on, Mr. Zhao. I'll call the president's office now."
The clerk picked up the phone on the desk and dialed a number. Soon, she hung up and said, "Mr. Zhao, please come with me."
A proud smile formed on Zhao Kai's face. Finally, Lin Momo had agreed to see him. He looked around and strolled to the elevator with the clerk.
Mancherk Company was a giant in the global pharmaceutical sales industry. A newly-founded Chinese medicine company couldn't afford to offend them.
The other representatives didn't protest at all. Mancherk Company was too famous for them to even feel envy.
Qin Haodong entered the elevator as soon as Zhao Kai had walked in. Nobody stopped him, as he was the boss of the company. He walked straight into Lin Momo's exclusive elevator.
The other medicinal representatives, who were irritated, protested to the front-desk clerk, "Who's that guy? How can he just walk in there? Shouldn't he have made an appointment?"
"Yeah, the representative of Mancherk Company walked in and we said nothing because his company's huge and we are no match for him. What about this guy, though?"
The clerk glanced at them and smiled before she explained, "That's the boss of our company, the inventor of the Blood Revival Elixir. What do you think? Does he need to make an appointment?"
The representatives were shocked and regretful. They had come by to visit Lin Momo, but they had forgotten about the other boss, Qin Haodong.
They regretted it a lot, because they could've won the dealership of the medicine if they had bonded with Qin Haodong. They had just missed this opportunity.
No wonder they hadn't recognized Qin Haodong. Every time he showed up in public, he used some magic on his face, making it slightly different from how he looked in photos. The slightest change made the photos look like him but not fully match his appearance.
Zhao Kai followed the clerk and entered Lin Momo's office. He didn't greet her before he sat directly on the sofa across the room. Then, he raised his chin, checked out Lin Momo, and introduced himself. "You're Lin Momo, right? I'm the head of Mancherk Company's Celestial Empire Branch. I'm here today to discuss the dealership of the Blood Revival Elixir with you."
Lin Momo frowned. Mancherk Company was the largest drug chain company in the world. She currently had no plans to sell the Blood Revival Elixir to the rest of the world, but she knew she would have to enter the international market someday. That was why she met Zhao Kai without an appointment. She had not known that the guy would be so arrogant. It looked like he looked down upon Tangmen Company a lot.
"Mr. Zhao…"
Lin Momo was immediately interrupted by Zhao Kai before she could open her mouth.
Zhao Kai said proudly, "Mancherk Company would be glad to represent your products provided that we can be the rule-makers. You have to accept three requests from us, or I'll just pass.
First, you will stop selling the Blood Revival Elixir in the Celestial Empire now. Have you lost your mind? 680 yuan for a box of meds with such a high value? That's basically charity.
Second, after we sign the dealership contract, we'll share the profit thirty-seventy, which means 30% of the profit will go to you and 70% to us.
Third, the right to adjust the price of the Blood Revival Elixir shall belong to us. We'll make it as high as we want."
Lin Momo was shocked by this arrogant guy. Who the hell was he? God? Those were three tough requests. She couldn't possibly accept even one of them, let alone three.
Thus, she asked, "Mr. Zhao, are you having a hangover? Did you not have enough sleep this morning? Or did you forget to take your meds at home?"
"What do you mean?" Zhao Kai, who was pissed, asked, "You found that harsh, didn't you? Don't forget that we're Mancherk Company. We're totally different from those small companies. Cooperating with us would bring you big advantages.
As the president of the group, you should be more far-sighted and able to see the truth. No matter how great the Blood Revival Elixir is, it's a kind of Chinese medicine. Have you seen the miserable lots of Chinese medicine in Europe and America nowadays?
Many developed countries in Europe and the United States believe that Chinese medicine is unscientific. No one believes in Chinese medicine, and no one believes it cures diseases. Your company is too small to make its own way into the European and American markets. You will never get this done unless Mancherk helps you with its great influence.
Moreover, Mancherk has a wide sales net around the world, and we have the best sales marketing team. If you hand the dealership of the Blood Revival Elixir to us, you can quit worrying about sales. All you'll need to do is stay at home and count the money flowing in."


text:
I've said it before, and I'm sure it'll be true again. About the time I think I know about all of the editions of Robert McCammon's books that have been published around the world, I will stumble over something I never knew existed. This time: books in Thailand! It seems that Thai publishers produced a Thai translation of Baal at some point, as well as two different printings of an anthology called The Creep in the Night, which contained a Thai translation of the short story "Nightcrawlers." You can see the covers below and in the Book Cover Gallery. Click on a cover to see a larger version of it.
One brand-new book is from Turkey: Gölge Oyunu is a Turkish trade paperback translation of Shadow Show: All-New Stories in Celebration of Ray Bradbury. That anthology included Robert McCammon's "Children of the Bedtime Machine."
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text: Hello! August has been a pretty full month for us around here - four birthdays (three all in the same week!), my sisters participating in the Bible Bee for the first time, a baby shower, a graduation, pea picking, and my most recent explorations into the world of brewing kombucha (which has been SUPER fun!).… Continue reading A Peek Into My Life: August Birthdays, Bible Bee, Baby Shower & Kombucha


text: The US Department of Agriculture estimates the value of field crops, fruits and nuts, and commercial vegetables produced on the 2 million farms across the US at more than $87 billion a year. Although that may seem like a staggering sum, profit margins for individual farmers are typically very slim. As a result, farmers are likely to take seriously any technological advance that will help increase those margins. Much of the farming community is adopting "precision farming": tailored methods of seeding, irrigating and treating specific fields in the most efficient manner. Global Positioning System locating, site-specific soil sampling and harvest yield monitoring are becoming accepted tools in the field. Now optical remote sensing systems are poised to join these tools to improve farming efficiency in the next century.


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text: Loved every minute inside the hotel! Gorgeously designed, comfortable, surrounded by amazing views. Even with a street view room I was lucky enough to face the hills covered in white! Room was big, with a comfortable bed also big enough for us. Breakfast was great, delicious pastries (especially the cheese cones) accompanied by friendly staff and nice ambient music. And unique views of the lake and mountains. Front desk staff was always friendly, warm and helpful. Terra Restaurant had delicious gourmet choices, fair price with great wine selections, from the cheapest to the priciest. Pool was nice, I was unfortunate to encounter some badly behaved children who dangerously jumped into the water non stop and I couldn't relax for long. I thought the water was a bit chilly also. It's very close to the town center, but it's only downside is that the sidewalk isn't made of concrete and after snow or rain the ground turns into a slippery mud (without appropriate boots I'd surely have slipped many times). If the sidewalks were fixed I'd give this hotel a paradise on earth status!


text:
A-Z List of Databases
Displaying 10 of 20 Items.
The database provides article-level access for thousands of e-journals available through EBSCO Subscription Services (EJS).
current; start dates vary depending upon title
Full-text reproductions of books, pamphlets, and broadsides published in the United States and American colonies from 1639 to 1800.
1639 - 1800
Full-text reproductions of 36,000 American books, pamphlets and broadsides published in the United States from 1801 to 1819.
1801 - 1819
The database includes specialized, editorially-curated A&I resources studying the critical issues affecting Earth's air, land, and water environments.
1950 -
Ebook Central (ProQuest) is an electronic book collection that spans all academic subject areas. Users can search for eBooks by title, author, subject, ISBN, LC call number, etc.
More than 4000 full-text electronic books in education, psychology, science, business, economics, computer science, history, and reference works that have been purchased for the CSU eBook Pilot Projec
EconLit, the American Economic Association's electronic database, is the world's foremost source of references to economic literature.
The Economist Historical Archive ('EHA') is the fully searchable complete facsimile edition of The Economist, the weekly paper which is essential reading for anyone engaged in politics, curr
1843 - 2015
Education Database supports the study and application of education across all levels of education, including early childhood education, primary and secondary education, and higher education.
1988 -
Education Full Text™ provides full text of articles from over 350 journals as far back as 1996, in addition to indexing of more than 770 periodicals dating back to 1983.
Full-Text 1996 - present, Indexing 1983 - present


text:
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COVID NOTICE: We're working to ship orders ASAP. Be advised that shipping/delivery delays are possible and may come without notice. NORMAL US MAIL/POSTAL SERVICE DELIVERY ESTIMATES NO LONGER APPLY. We recommend using UPS services to expedite shipping, however UPS has changed their delivery guarantees as well.
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4 yards, but the price is really good, 80 percent of public code for meritorious service. GT-2000 2 is positioned on the top level support. While not as much as Kayano, but more suitable for medium weight runner. GT-2000 although not as Kayano a long series, but popularity is not bad, since 1995 the early GT-2010 in technology uses Gel cushioning material of plastic, EVA midsole, DuoMax dual density anti-tilting device and other key technologies, with the development of technology, the latest in the end GT-2000 2 uses double buffering FluidRide, foot stability Guidance Trusstic System, Dynamic dual anti-tilting device for Dynamic density DuoMax, as well as main Palm suspension Gel before and after (the forefoot part using hidden design) allows you to see the elements of science and technology become more and more rich.
Plus size fringe benefits: ASICS stability GT-2000 2 BR top men shoes $56.53


text:
By Richard Brewer
Land trusts, or conservancies, shield land by means of possessing it. This version of saving land via deepest motion has turn into dominant purely because the Eighties and this booklet bargains a whole remedy of the U.S. land belief circulate as a vital function of present efforts to guard the surroundings.
Read Online or Download Conservancy: The Land Trust Movement in America PDF
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What Every Engineer Should Know About Business Communication (What Every Engineer Should Know)
Engineers needs to own a number company verbal exchange abilities that let them to successfully speak the aim and relevance in their proposal, procedure, or technical layout. This precise company conversation textual content is filled with sensible suggestion that might increase your skill to— · marketplace principles · Write proposals · Generate enthusiasm for study · bring shows · clarify a layout · manage a undertaking crew · Coordinate conferences · Create technical experiences and requirements concentrating on the 3 severe verbal exchange wishes of engineering professionals—speaking, writing, and listening—the ebook delineates serious communique recommendations required in lots of crew settings and paintings events.
Science and Public Policy: The Virtuous Corruption of Virtual Environmental Science
This ebook argues that the digital nature of a lot environmental technology and the applying of non-science ideas comparable to the precautionary precept facilitate the virtuous corruption of environmental technology. Drawing upon examples from conservation biology and variety Aynsley Kellow illustrates that the matter is extra common than this region by myself may recommend and is usual within the vital box of weather technological know-how.
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Dealing with typical assets sustainably is a fancy activity that calls for the involvement of many various stakeholders. community preparations are more and more used to aim and attain such sustainable administration. This booklet assesses the perform of such networks utilizing unique examine into case stories of panorama, habitat and water administration from England, Norway, Sweden, Spain and Zimbabwe.
The Invasion of Indian Country in the Twentieth Century: American Capitalism and Tribal Natural Resources
The fight among Indians and whites for land didn't finish at the battlefields within the 1800s. whilst this antagonistic period closed with local american citizens compelled onto reservations, not anyone anticipated that wealthy traditional assets lay underneath those lands that white the USA could desperately hope. but oil, bushes, fish, coal, water, and different assets have been found to be in nice call for within the mainstream industry, and a brand new struggle started with Indian tribes and their leaders attempting to guard their tribal usual assets in the course of the 20th century.
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Additional resources for Conservancy: The Land Trust Movement in America
Sample text
The proposal for forming the Trustees of Reservations came from the merger of his profession with his feeling of duty toward society. S. population was rural, and no city was as large as 400,000. By 1890, the strictly rural percentage had dropped to 65 percent, and the largest city, New York, had a population of nearly two million. Accounts by some historians and sociologists suggest a picture of the rural areas emptying as men and women sought jobs in the factories, mills, and sweat shops of the cities.
31 Even though the Trustees was immediately successful, few new land trusts were started. Instead, advocacy organizations, some of which occasionally owned land, were forming throughout the East in the years prior to 1900. Besides the Audubon societies and a couple of Ohio museums (Cincinnati and Dayton), only one organization listed in the Land Trust Alliance's one line short Brewer: Conservancy page 25 History 25 directory had a pre-1900 founding date. This was the Connecticut Forest and Park Association, founded in 1895.
The League also had an executive committee of about twenty members, chaired by Merriam. We have seen that the lands available for protection by the Trustees of Reservations were privately owned, relict fragments, while the Sierra Club targeted large blocks still in the hands of the federal government. Large stands of coastal redwoods north of San Francisco Bay, the focus of the Save-the-Redwoods League, still existed, but the government had already given the land away. The groves were falling to the lumberman's ax at an increasing rate and, realistically, most could only be protected by purchase.


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at 02:15, 11-25-2020.
Kirkland Area Attractions
Gunnar Nordstrom Gallery
Howard/Mandville Gallery
Argosy Cruises
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A shocking moment took place in Hangzhou, a city in east China on July 2. A two-year-old girl named Niu Niu fell from a 10th floor building and was caught by a "super-mother" with her bare arms.
The super-mother's name is Wu Juping,32- year-old. The accident happened at around noon while Niu Niu's grandmother was outside to bring in quilts. The little girl woke up from her nap and her grandmother was nowher to be found. She then climbed onto the windowsill trying to find her grandmother.
A neighbor from the opposite building saw Niu Niu's body hanging out the window. A resident from the ninth floor tried to reach Niu Niu by using a ladder, but failed.
Wu Juping kicked off her high-heeled shoes, reached the falling child in time to break the toddler's fall, preventing the girl from hitting the ground at full force.
Wu and Niu Niu were immediately sent to a local hospital. Doctors said that Niu Niu suffers from multiple organ damage and is still in a deep coma.
"It's luck in the midst of sadness. It's miracle Niu survived the accident," Said Dr. Xu of Zhejiang Children Hospital.
The brave lady Wu Juping, an employee of Alibaba, became a mother seven months ago. Her left arm was severely injured in the accident. She woke up at 4 am the second day, and couldn't sleep well at all.
The story soon spread online, complete with photographs of the scene, describing the incident and Wu's heroic efforts. Thousands of people were moved by her bravery and prayed for her and the child to recover soon.
Wu said, "I myself have a seven-month-old baby, who happened to fall off a chair and hurt her lips, they bled a lot. So I thought if this little girl fell off from the 10th floor, the consequences might be horrible. So I thought I should help her."
"I am totally flattered. I just did a normal thing that everyone else would've done if they came across the same thing. It's so unexpected that people care about me so much and I would never thought to be known through this action," said Wu.
Niu Niu's father said, "Today is the first time I have felt such warmness in my heart."
According to the doctor, Wu Juping has a comminuted fracture in left arm, and an operation is needed. Many believe that a mother who was willing to sacrifice so much for a complete stranger should be cherished by society.
(China.org.cn by Li Jingrong July 6, 2011)


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1. 2018 Audi A1
Since its launch in 2010, the Audi A1 did not make much of the premium supermini segment. There was something darkened by the rival MINI. However, the second generation A1 is set to lock horns with its iconic British rival. Priced at $ 18,000, 2018 The Audi A1 comes with high-tech makeup, including a newly created infotainment setting and plush trim. Based on the VW group's new MQB architecture, it should be fun to drive. Spy images show a more hatchback-like design as opposed to the existing model's bubble shape. The release date is expected in early 2018.
This small city car will have a wide range of engines. In addition to 1.0-liter and 1.5-liter engines, it also comes with a 1.6-liter diesel engine. Some rumors say that in 2019, Audi will offer an S1 version powered by a 2.0-liter turbocharged petrol engine.
2. 2018 BMW i8 Roadster
One of BMW's long-awaited models, the i8 Roadster is clearly unveiled in 2018. The coupe will have a hybrid powertrain. At about $ 155,000, it is not meant for the weak hearts. It is unclear whether BMW should use lighter fabric or go completely open for this. Spy images of the prototype hint at threatening front design.
This convertible BMW Roadster will be powered by a three-cylinder 1.5-liter engine. This motor in tandem with electric motor provides fantastic 362 hp. The release date for the i8 Roadster is confirmed for 2018.
3. 2018 Audi Q8
A large sized SUV SUV is fashionable and everyone from Bentley to Rolls Royce is set to reveal their contenders. Of course, Audi does not want to be left. Q8 is the upcoming flagship SUV from the company and it will have an expected price tag of $ 88,000. It will have a powerful hybrid engine and sumptuous interior. The huge grill and the aggressive exterior styling make it quite wonderful.
As for the specifications, this Audi large SUV currently has only 3.0 liters of TFSI engine, which in the tandem with electric motor provides 444 hp. In addition to this powertrain, we can expect in the future a 4.0-liter V8 turbo diesel engine and probably a petrol engine.
The release date od Audi Q8 is still unconfirmed, but we can certainly say that it will be available for purchase in 2018.
BMW has prepared a number of new models for the launch of 2018, but the iconic German car manufacturer is rebuilding current popular models! Spy images of the new 2018 BMW X3 have emerged and they indicate that the new model will be built on the brand's highly tense modular UKL platform. It is expected to be lighter than the existing model due to heavy use of high-strength steel and aluminum. The expected price tag for the 2018 BMW X3 is $ 42,000.
The BMW SUV will have three powertrain options in the offer. The basic model comes with a 2.0-liter turbocharged diesel engine. This engine has 180 hp and 280 lb-ft of torque. The second option is also the 2.0-liter engine, but with 240 hp. The most powerful engine in the new 2018 BMW X3 is a 3.0-liter turbocharged 300 hp diesel engine.
The release date is expected during the third quarter of this year.
5. Audi Q6 e-tron
The first electric SUV from Audi will not be a variant of the regular Q6. It will definitely be worn during Q7. The design's similarities with the 2015 e-throne Quattro concept presented at the Frankfurt Motor Show would be there. The approximate price would be close to $ 75,000. It is expected to offer rapid acceleration even though there is no fuel-based drive unit. The exterior style will be like a sporty SUV.
This electric SUV will have two versions that have the same battery capacity of 100 kWh. The first will have 303 hp and the other will have 408 hp. Autonomy of both versions should be 185 to 220 miles.
Audi has announced that the production version of the Q6 e-tron will be ready for 2018.
The upcoming Z4 is among the most anticipated models of BMW that will be released in 2018. Expected to be priced at $ 38,000 Z4 is aimed at the Porsche 718 Boxter. The model will have a soft top. It will have dual seat design and few rear wheel drive.
The new 2018 BMW Z4 will be offered with two engine options. The first will be a 2.0-liter 4-cylinder engine with 250 hp. The second option would be a 3.0-liter, 6-cylinder, 335-hp engine.
The release date for this BMW roadster is expected in 2018.
7. 2018 Skoda Yeti
The Yeti, Skoda's popular SUV is all for its 2nd generation debut. The prototype model has been tested several times. Design tracks from their siblings Kodiaq are expected to be there in the last model. The boxing building of the current model will not be there and it will be built on the MQB platform. Changes in wheel design and grille are also expected. The base trim price would be around $ 20,000.
For engine units, we expect power transmissions from the VW game, including a 1.0-liter 3-cylinder turbine petrol and 1.6 and 2.0-liter TDI diesel engines. Because hybrid variant is also possible, it can combine 1.4-liter petrol engine and electric motor.
The new Yeti is expected in showrooms from the beginning of 2018, at a starting price of about $ 18,000 for the base version.
8. 2018 Toyota Supra
Toyota Supra makes headlines because it's a little different than other cars made by the Japanese car race. Toyota is developing this sporty coupe in alliance with BMW. Some design elements in the previously presented Toyota FT-1 concept can be found in the last model. Details of the driveline are not clear but the car can be priced around $ 62,000.
As we discovered in 2018, the Toyota Supra will be powered by a 6-cylinder, 3.0-liter turbo engine connected to a hybrid engine that provides a total power of 470 hp.
This model will be available in the sale from January 2018.
9. 2018 Volkswagen Touareg
Touareg is one of the most popular models from VW and it will be a makeover for 2018. It will share the Bentley Bentayga and Audi Q7 platform. So, 2018 Touareg will have a light body. At first appearance it is similar to the Audi Q7 to an extent. It is likely to be priced at $ 55,000.
Volkswagen has still not announced which drives provide the power to the new Touareg.
10. 2018 Mercedes A-Class
The Mercedes A class is set to get a makeover for 2018 and this is natural considering the steady popularity of the A series. Focused on the BMW X1, the A-class will get sharper exterior styling. This is among the more affordable Mercedes models you can buy and the starting price would be close to $ 25,000. A top of the line Mercedes-AMG A 45 variant can also be presented later. Everything from the infotainment attitude to the quality of the cab upholstery will increase.
The old 2.1 liter diesel engine will be replaced with new 2.0 liters with 160 hp and 190 hp.
The release date is expected in early 2018, even until the end of next year, you can expect Mercedes A-Class since the release.
These are ten cars from different categories that will be completely redesigned. As you can see, this article contains all categories from small city cars to Sport Utility Vehicles.


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Emphasizing the integration of mathematical expressions with clear physical associations, this challenging graduate-level textbook on convective heat and mass transfer reviews the laws of thermodynamics and fluid motions, behavior of laminar and turbulent flows in a variety of conditions, natural free convection in space, and flows through porous media.
"synopsis" may belong to another edition of this title.
An inspiring and unconventional engineering text on the frontiers of science, the first edition of Convection Heat Transfer quickly established itself as a premier professional reference, one that offered an entirely new and invigorating approach to research and teaching. Updated and revised to reflect the field's latest research and technical innovations, the Second Edition is an authoritative practical reference and basic graduate-level text to convection heat transfer. Like its predecessor, the new edition balances traditionally rival corners of convection teaching and research, such as forced and natural convection, laminar and turbulent flows, fluids and fluid-saturated porous media, as well as theory and empiricism. Yet the new edition also focuses on the growing concerns in energy and ecology, with an emphasis on man's enhanced role in living equitably with nature. The book's unique topical breadth and arrangement, the first in a text on heat convection, reflect today's environmental realities:
The up-to-date edition of the bestseller on convection heat transfer
A revised edition of the industry classic, Convection Heat Transfer, this Third Edition chronicles how the field of heat transfer has grown and prospered over the last two decades. Students will find this new edition more accessible, while not sacrificing its thorough treatment of the most up-to-date information on current research and applications in the field.
One of the foremost leaders in the field, Adrian Bejan has pioneered and taught many of the methods and practices commonly used in the industry today. He continues this book's long-standing role as an inspiring, optimal study tool by providing:
Complete with hundreds of informative and original illustrations, Convection Heat Transfer, Third Edition is the most comprehensive and approachable text for students in schools of mechanical engineering.
"About this title" may belong to another edition of this title.
Book Description Wiley-Interscience. Condition: new. Seller Inventory # think0471896128
Book Description Condition: New. New. Seller Inventory # M-0471896128
Book Description Condition: New. New. Seller Inventory # EX-0471896128
Book Description Wiley-Interscience, 1984. Hardcover. Condition: New. Brand New!. Seller Inventory # VIB0471896128
Book Description Condition: New. Seller Inventory # XAY--096


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I've dropped the ball on D&D for far too long. Let's get back to it. We're reading The Gathering Storm by Adrian Bott, for D&D 3.5, with an eye to running it using Halberds & Helmets. Chillhame is the first adventure, part two, where the player characters get to explore some stuff underground.
One hook for the part are missing children. Who doesn't want to rescue missing children? As a player, I recognise a plot hook when I see one. There's also John's tomb, which might possible have his magic sword. The tomb has a missing words puzzle. I'm not so sure how I'd run it. In D&D 3.5 you would use the Decipher Script skill. But taking your time to carefully clean the tombstone also reveals everything. Not much of a puzzle, I guess? I like the thought, though.
There are two hobgoblin tribes in the area and there's signs of combat near the tomb. This is good. One thing leads to another. With various skills checks players can discover the thing you want them to discover anyway: two trails, a woodland trail and a hill trail.
The woodland trail leads to a hobgoblin camp. The hobgoblins drawings are cool, they look like tattooed punk biker hobgoblins. There are traps guarding the camp, so it pays to approach it carefully. This is the camp of Split Ear and his eight hobgoblins, plus some non-combatant hobgoblins (children). I wonder whether I'd use them. Are hobgoblins mythical monsters, grown from rotten trunks and mud pits, or are they simply humans under another name? I don't know. I think I'd run them as tattooed punk biker humans. And so I'll have to make plans for players trying to resolve the situation: the hobgoblins are acting like bandits, do the get hanged? What about the hobgoblin children? Is there at least the option of settling them somewhere? Giving them control over the old mill after kicking out Jim Oakenbough? This is going to be a tough one.
Once the players go to the mine, I like how Jim Oakenbough shows up again and tries to intimidate them. And if you return from the mines, he'll try to kill you.
And with that we get to the mines!
The mines are under the control of the second hobgoblin tribe, the Talks-with-Fists band. There are side entrances, a grate covering a ventilation shaft, a test shaft, there are tracks to find, and empty bottles in the buildings outside. I like how there's a hobgoblin drunkard right at the beginning who can be questioned.
The mine has a mine cart that works! Yay!
I don't like how some of the references here are wrong. The ventilation shaft ends in area 4 on the map but the text says area 6. The mine cart says that the path it takes depends on the setting in area 7, which say south, which the map says is area 9, but the text says area 5. What is going on, here? But worse: those extra entrances don't help the party sidestep anything. That's why I'd ignore them.
Similarly, I don't think there's much of a point to the spider encounter. Drop it.
So here's I think what we need:
I'm not sure how to explain the shaft, though: the hobgoblins on the lower level seem to have no way back up unless somebody pulls them up in the lift? That's not a smart position to be in. I'd probably add at least a ladder inside the shaft and maybe the lift is permanently broken, lying down there, smashed?
Anyway, at this point we have three things pointing to the next level: the remaining hobgoblins, the kids, and the ghost.
The second level of the dungeon is flooded mine tunnels, which is excellent visuals as anybody who has seen Aliens knows.
Down here are two hobgoblins standing guard in 13d which isn't on the map. Oh well. And then there's the boss, with two more hobgoblins, and some hobgoblin children.
Finally, two things to discover: diving under water to find the last child in a cul-de-sac, and the weakened wall that allows players to break through into the actual underworld. There's Grobni the Surveyor, a duergar.
I wonder how to run a duergar in combat with their ability to enlarge themselves. Do I recompute the stats on the fly? How did the old editions handle that. In the AD&D monster manual it says they can also enlarge themselves. Weird.
Returning to the surface and warning the village of the invasion to come is cool but I think we'd also need to add a bit of dungeon material just in case the players go investigating. They need to see more than just a door that's magically locked. How did Grobni expect to get back? Can't he open the door? I'm sure he can! The players need to see those duergar barracks, I think.
Well, and there you have it. We've reached pages 45 and 46 with a discussion of the aftermath: how the villagers are going to react, how to push them to evacuate, how to proceed if the players are going to warn the rest of the kingdom, and so on.
I think at this point I should have a pretty good plan set up on how to drive the players before the army if they refuse to do what the text implies: take the evidence to Saragost and warn the villages along the way.
That's the next chapter: Raising the Alarm.
Tags: The Drow War Old School RPG


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Show me a picture of a porcupine
How Do You Hug a Porcupine? by Laurie IsopThe third book in the General Mills Spoonful of Stories series!
Can you imagine hugging a porcupine? Sure, its easy to picture hugging a bunny or even a billy goat, but where would you begin to try to hug a porcupine?
After seeing all his friends hug their favorite animals, one brave boy works up the courage to hug a porcupine, but the porcupine isnt so sure he wants to be hugged!
The latest contest winner Laurie Isop and illustrator Gwen Millward arent afraid to ask the pointed questions!
Facts About Porcupines
Discussion in ' Somali Politics ' started by Bohol , Jul 5, Log in or Sign up. Somali Spot Forum, News, Videos. So this morning the Porcus tried their usual ambush attempt against a already armed SY militia and you know how the results turned out. Several dead Porcus and they run away from their injured. This incident originally started after the Porcus tried to touch the grass of the area for livestock.
Sharp quills
Set it all free lyrics ~ Scarlett Johnsson
The porcupine and hedgehog are prickly mammals. They are often confused because they both have sharp, needle-like quills on their body. Porcupines and hedgehogs differ in size, defensive behavior, diet and country of habitation. Their quills also have different features. Porcupines and hedgehogs differ in size and the structure and function of their quills.
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Thomas Paine Quotes in Common Sense
The cause of America is in a great measure the cause of all mankind. Many circumstances hath, and will arise, which are not local, but universal, and through which the principles of all Lovers of Mankind are affected, and in the Event of which, their Affections are interested. The laying a Country desolate with Fire and Sword, declaring War against the natural rights of all Mankind, and extirpating the Defenders thereof from the Face of the Earth, is the Concern of every Man to whom Nature hath given the Power of feeling; of which Class, regardless of Party Censure, is the AUTHOR.
Some writers have so confounded society with government, as to leave little or no distinction between them; whereas they are not only different, but have different origins. Society is produced by our wants, and government by our wickedness; the former promotes our happiness positively by uniting our affections, the latter negatively by restraining our vices. The one encourages intercourse, the other creates distinctions. The first is a patron, the last a punisher.
In short, monarchy and succession have laid (not this or that kingdom only) but the world in blood and ashes. 'Tis a form of government which the word of God bears testimony against, and blood will attend it.
The sun never shined on a cause of greater worth. 'Tis not the affair of a city, a country, a province, or a kingdom, but of a continent— of at least one eighth part of the habitable globe. 'Tis not the concern of a day, a year, or an age; posterity are virtually involved in the contest, and will be more or less affected, even to the end of time, by the proceedings now. Now is the seed time of continental union, faith and honor. The least fracture now will be like a name engraved with the point of a pin on the tender rind of a young oak; the wound will enlarge with the tree, and posterity read it in full grown characters.
We may as well assert that because a child has thrived upon milk, that it is never to have meat, or that the first twenty years of our lives is to become a precedent for the next twenty. But even this is admitting more than is true, for I answer roundly, that America would have flourished as much, and probably much more, had no European power had any thing to do with her. The commerce, by which she hath enriched herself are the necessaries of life, and will always have a market while eating is the custom of Europe.
Europe, and not England, is the parent country of America. This new world hath been the asylum for the persecuted lovers of civil and religious liberty from every part of Europe. Hither have they fled, not from the tender embraces of the mother, but from the cruelty of the monster; and it is so far true of England, that the same tyranny which drove the first emigrants from home, pursues their descendants still.
But if you say, you can still pass the violations over, then I ask. Hath your house been burnt? Hath your property been destroyed before your face? Are your wife and children destitute of a bed to lie on, or bread to live on? Have you lost a parent or a child by their hands, and yourself the ruined and wretched survivor? If you have not, then are you not a judge of those who have. But if you have, and still can shake hands with the murderers, then are you unworthy the name of husband, father, friend, or lover, and whatever may be your rank or title in life, you have the heart of a coward, and the spirit of a sycophant.
Wherefore, her own interest leads her to suppress the growth of ours in every case which doth not promote her advantage, or in the least interferes with it. A pretty state we should soon be in under such a second-hand government, considering what has happened! […] And in order to shew that reconciliation now is a dangerous doctrine, I affirm, that it would be policy in the king at this time, to repeal the acts for the sake of reinstating himself in the government of the provinces; in order, that HE MAY ACCOMPLISH BY CRAFT AND SUBTILTY, IN THE LONG RUN, WHAT HE CANNOT DO BY FORCE AND VIOLENCE IN THE SHORT ONE. Reconciliation and ruin are nearly related.
But where says some is the King of America? I'll tell you Friend, he reigns above, and doth not make havoc of mankind like the Royal Brute of Britain. Yet that we may not appear to be defective even in earthly honors, let a day be solemnly set apart for proclaiming the charter; let it be brought forth placed on the divine law, the word of God; let a crown be placed thereon, by which the world may know, that so far as we approve of monarchy, that in America THE LAW IS KING.
O ye partial ministers of your own acknowledged principles. If the bearing arms be sinful, the first going to war must be more so, by all the difference between wilful attack and unavoidable defence. Wherefore, if ye really preach from conscience, and mean not to make a political hobby-horse of your religion, convince the world thereof, by proclaiming your doctrine to our enemies, for they likewise bear ARMS.