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Turnovers Doom Chargers, Vikings Cruise
by Anthony Galler · December 16, 2019
The Matchup
The Minnesota Vikings traveled to the west coast for a showdown with the Los Angeles Chargers on Sunday. This game was initially scheduled for Sunday Night Football but was flexed out in favor of Bills versus Steelers. Minnesota aimed to stay one game back of Green Bay in the standings, before their week 16 meeting next Sunday.
This game marked the first game back for wide receiver Adam Thielen, who returned from a hamstring injury. Sunday's matchup was Minnesota's away finale for the 2019 season. A win would give them a 4-4 road record and a 10-4 record overall. Could the Chargers pull off an upset, or would Minnesota cruise to a victory?
The Vikings wasted no time getting on the scoreboard. On the game's opening possession, they marched down the field for a touchdown drive, capped by a 1-yard TD pass from Kirk Cousins to rookie Irv Smith Jr. This was Smith Jr's second touchdown of the year, and the only touchdown pass thrown by Cousins on the afternoon. Dan Bailey failed to convert the PAT, but the Vikings still lead 6-0.
Chargers kicker Michael Badgley tacked on a 41-yard field goal a few minutes later, and Minnesota took a 6-3 lead into the second quarter.
Another Dan Bailey field goal gave Minnesota a 9-3 advantage, but the Chargers would answer back. Philip Rivers took his team on a ten-play, 75-yard drive, which culminated in a 2-yard touchdown strike to wide receiver Mike Williams. The Chargers now lead 10-9, but not for long.
The Vikings later retook the lead 12-10, but the Chargers offense had one last opportunity to score before halftime. Los Angeles was on the edge of field goal range with time ticking down in the second quarter, and then disaster struck.
Philip Rivers took a sack and lost control of the football. The ball was picked up by Vikings lineman Ifeadi Odenigbo and returned 56 yards for a defensive score. The Phillip Rivers sack fumble was one of SEVEN turnovers for the Los Angeles offense on the day. The Vikings cruised to a 19-10 lead into the locker room, despite just one offensive touchdown.
The Chargers fumbled yet again on their opening possession of the third quarter. Minnesota failed to score on their next drive, but that was the least of their worries. Star running back Dalvin Cook took a hard fall on the same shoulder that he injured two weeks ago in Seattle.
Cook managed to shake off the previous injury, but this one appeared to be more serious. He immediately left the game for the blue medical tent and was declared out of the game just minutes later. Cook's status has not yet been announced, but all eyes will be centered on him as the Vikings head into week 16.
The rest of the third quarter proved to be rather uneventful for both sides. The Vikings added two additional field goals from Dan Bailey, while the Chargers offense was shut out. Minnesota held a 25-10 advantage at the end of three quarters.
Boone goes Boom
The fourth quarter became the Mike Boone show in Dalvin Cook's absence. Backup running back, Alexander Mattison did not suit up for this game due to an ankle injury, so it was Mike Boone handling tailback duties. Naturally, Minnesota ran the ball for much of the fourth quarter with a big lead already. Boone rushed for a pair of scores in the fourth quarter alone and finished his day with 13 carries for 56 yards and two touchdowns. It wasn't a bad afternoon for a guy who was the Vikings third-string runner just a week ago.
Meanwhile, the Chargers offense continued to be pitiful throughout the fourth quarter. Los Angeles committed turnovers on their final three possessions of the game. This included two more interceptions for Phillip Rivers, who now has 18 picks this season. The Vikings defense shut out the Chargers in the second half and cruised to an eventual 39-10 victory.
Image via: https://www.dailynorseman.com/
The Chargers dreadful season reached new heights on Sunday with a seven-turnover performance. After a wild card berth a year ago, Phillip Rivers and the rest of the offense looked lost. Los Angeles drops to 5-9, and are now guaranteed a losing 2019 season. On the other hand, the Vikings improved to 10-4, while staying one game back of Green Bay in the NFC North. Next week's showdown will likely be for all the marbles when the Vikings and Packers meet once again.
Green Bay won the first matchup 21-16 in week 2, but the Vikings will be at home this time. Minnesota got Adam Thielen back this week, though the status of Dalvin Cook will now be on everyone's minds throughout the week. Monday Night Football will be the perfect stage for these teams in week 16, as the NFC North crown is up for grabs.
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Laninamivir octanoate (LO) is an octanoyl ester prodrug of the neuraminidase inhibitor laninamivir. After inhaled administration, LO exhibits clinical efficacy for both treatment and prophylaxis of influenza virus infection, resulting from hydrolytic bioactivation into its pharmacologically active metabolite laninamivir in the pulmonary tissue. In this study, we focused on the identification of LO-hydrolyzing enzymes from human pulmonary tissue extract using proteomic correlation profiling—a technology integration of traditional biochemistry and proteomics. In a single elution step by gel-filtration chromatography, LO-hydrolyzing activity was separated into two distinct peaks, designated as peak I and peak II. By mass spectrometry, 1160 and 1003 proteins were identified and quantitated for peak I and peak II, respectively, and enzyme candidates were ranked based on the correlation coefficient between the enzyme activity and the proteomic profiles. Among proteins with a high correlation value, S-formylglutathione hydrolase (esterase D; ESD) and acyl-protein thioesterase 1 (APT1) were selected as the most likely candidates for peak I and peak II, respectively, which was confirmed by LO-hydrolyzing activity of recombinant proteins. In the case of peak II, LO-hydrolyzing activity was completely inhibited by treatment with a specific APT1 inhibitor, palmostatin B. Moreover, immunohistochemical analysis revealed that both enzymes were mainly localized in the pulmonary epithelia, a primary site of influenza virus infection. These findings demonstrate that ESD and APT1 are key enzymes responsible for the bioactivation of LO in human pulmonary tissue.
Laninamivir octanoate (LO), an octanoyl ester prodrug of the neuraminidase inhibitor laninamivir, demonstrates a long-lasting antiviral effect in contrast to oseltamivir and zanamivir, and is currently used in clinical practice in Japan for treatment and prophylaxis of influenza virus infection. In confirmatory clinical trials, a single inhalation of LO demonstrated a therapeutic efficacy in both adult and pediatric patients (Sugaya and Ohashi, 2010; Watanabe et al., 2010; Watanabe, 2013). In addition, LO was recently reported to be efficacious in the postexposure prophylaxis of influenza in household contacts (Kashiwagi et al., 2013).
Hydrolytic activation of LO into the active form laninamivir in the pulmonary tissue is a critical factor for exerting its in vivo pharmacologic effect. After a single intranasal/intratracheal administration in mice and rats, LO was efficiently hydrolyzed to laninamivir, and then it was highly retained in the pulmonary tissue (Koyama et al., 2009, 2010) with a sufficient concentration in the epithelial lining fluids (ELF), a possible site of action of neuraminidase inhibitors (Koyama et al., 2013). In humans as well, after a single inhalation of LO (40 mg), laninamivir was generated and highly maintained in the ELF (Ishizuka et al., 2012), exceeding the in vitro 50% inhibitory concentrations for influenza viral neuraminidases over 10 days (Yamashita et al., 2009). A slow elimination of laninamivir was also observed in the systemic circulation, with an elimination half-life (t1/2) of approximately 3 days (Ishizuka et al., 2009; Yoshiba et al., 2011). These favorable pharmacokinetic characteristics are considered to result in the long-lasting effect.
In drug development, the prodrug approach has been widely used as a strategy for improving the physicochemical, biopharmaceutical, or pharmacokinetic properties of pharmacologically active agents, and approximately 10% of the drugs approved worldwide are reported to be classified as prodrugs (Rautio et al., 2008; Zawilska et al., 2013). Prodrugs require the bioactivation into their active forms by metabolizing enzymes to be therapeutically effective. On the other hand, the prodrug-activating enzymes may have a potential risk to influence interindividual variability in drug exposure and response, and possibly to induce drug-drug interactions. Therefore, it is very important to identify the prodrug-activating enzymes.
For the molecular identification of endogenous enzymes from biologic samples, biochemical purification has been used for decades as a general methodology, and a series of sequential separation by different chromatographic methods (ion-exchange, affinity, gel filtration, etc.) is usually required to achieve the purification. On the other hand, we have recently extended an advanced methodology named proteomic correlation profiling to identify drug metabolizing enzymes (Sakurai et al., 2013), whose basic concept was previously reported by Kubota et al. (2009). In this methodology, the biologic material is fractionated by column chromatography, followed by the calculation of each protein's correlation coefficient between the enzyme activity and proteomic profile of the fractions. Subsequently, possible enzyme candidates are chosen among proteins with a high correlation value, based on the fundamental assumption that protein quantity correlates with protein activity. This streamlined method enables us to require fewer purification steps with minimal starting material, and also to resolve the challenges associated with physicochemical stability and solubilization, as compared with the conventional biochemical purification. Therefore, the proteomic correlation profiling is considered to be a powerful and efficient tool for enzyme identification.
In the present study, from the human pulmonary tissue extract, we identified S-formylglutathione hydrolase (also known as esterase D [ESD]) and acyl-protein thioesterase 1 (APT1) as most likely candidates for LO-hydrolyzing enzymes, using the proteomic correlation profiling. We also performed further analysis to confirm the enzyme identification and to investigate the enzyme localization in the pulmonary tissue. We believe that this study can provide fundamental information on the LO bioactivation process in the target site.
Both LO and laninamivir were synthesized at Daiichi Sankyo Chemical Pharma Co., Ltd. (Tokyo, Japan) and their chemical structures are illustrated in Fig. 1. The internal standard (IS) of laninamivir ([2H3]laninamivir) was synthesized at Daiichi Sankyo Co., Ltd. (Tokyo, Japan). Palmostatin B was synthesized at Daiichi Sankyo RD Novare Co., Ltd. (Tokyo, Japan). The pooled human pulmonary subcellular fractions (S9, microsomes, and cytosol) used for LO-hydrolysis characteristics were obtained from XenoTech (Lenexa, KS), whereas the individual human pulmonary S9 (79-year-old, male) used for gel-filtration chromatography was obtained from Human and Animal Bridging Research Organization (Chiba, Japan). Formalin-fixed and paraffin-embedded human pulmonary tissue sections (62-year-old, male) were obtained from SuperBioChips Laboratories (Seoul, Korea). Ethical approval for these human specimens was obtained from the Research Ethics Committee at Daiichi Sankyo Co., Ltd., and Daiichi Sankyo RD Novare Co., Ltd. Diisopropyl fluorophosphate (DFP) and eserine were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and bis-p-nitrophenyl phosphate (BNPP) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Nacalai Tesque (Kyoto, Japan). Recombinant human esterase D (rESD) and acyl-protein thioesterase 1 (rAPT1) were obtained from Novoprotein Scientific Inc. (Shanghai, People's Republic of China). Rabbit anti-ESD and anti-APT1 antibodies were obtained from Sigma-Aldrich (St. Louis, MO) and Proteintech Group (Chicago, IL), respectively. All other reagents and solvents used were commercially available and were of extra-pure, guaranteed, or liquid chromatography coupled with mass spectrometry (LC-MS) grade.
Chemical structures of laninamivir octanoate (LO) and its active metabolite laninamivir. After inhaled administration of LO in humans, the active metabolite laninamivir is generated by an enzymatic hydrolysis of the octanoyl ester moiety. When dissolved in water, LO is equilibrated at 9:1 (3-acyl form:2-acyl form) and therefore IS defined as a mixture of the 3-acyl form (major) and the 2-acyl form (minor).
LO Hydrolysis Assay and Chemical Inhibition Properties.
For determination of LO-hydrolyzing activity in human pulmonary subcellular fractions (S9, cytosol, and microsomes) and recombinant proteins (rESD and rAPT1), the sample was incubated at 37°C for 15 minutes with 10 μM LO. The reaction was terminated by mixing with 2 volumes of acetonitrile. The sample was added an aliquot of IS and then filtered. The resulting sample was injected into the liquid chromatography coupled with a tandem mass spectrometry (LC-MS/MS) system consisting of API 4000 (Applied Biosystems, Foster City, CA) coupled to Shimadzu 20A (Shimadzu Corp., Kyoto, Japan). The analyte was separated on an analytic column, a PC HILIC Silica (5 μm, 2.0 × 150 mm; Shiseido Co., Ltd., Tokyo, Japan), with a gradient of 10 mM ammonium acetate and acetonitrile. The flow rate employed was 0.6 ml/min. The analyte was detected with an electrospray ionization (ESI) source operated in the positive-ion mode. MS data were acquired in selected ion multiple reaction monitoring mode using the mass transitions of m/z 347 → 60 and 350 → 60 for laninamivir and IS, respectively. The calibration curves were generated using the analyte to IS peak area ratio by weighted (1/x) least-squares linear regression over the concentration ranges of 1–1000 ng/ml (2.89–2890 pmol/ml). The enzymatic activity was expressed as a metabolite formation rate (pmol/min/mg) based on the production of laninamivir. For assay of chemical inhibition properties, each of the esterase inhibitors (DFP, BNPP, eserine, and DTNB) was added to the reaction mixture and further incubated with 10 μM LO. The results were expressed as percentages of the activity in the absence of inhibitors.
Human pulmonary S9 fraction (5 mg/ml, 20 mM HEPES; pH 7.0, 250 mM sucrose) was used as a starting material. The S9 fraction (500 μL) was incubated for 10 minutes, following the addition of 10% Nonidet P-40 (NP-40; 50 μL) and 5 M NaCl (50 μL). After centrifugation at 105,000g for 1 hour, the supernatant was filtered and loaded onto a gel-filtration column Superdex 200 (GE Healthcare UK Ltd., Little Chalfont, UK), which was equilibrated with running buffer (20 mM HEPES; pH 7.0, 150 mM NaCl and 0.05% NP-40) at a flow rate of 0.5 ml/min. The protein separation was performed with the same buffer at a flow rate of 0.5 ml/min, and the eluent was collected in a series of fractions at every 1 minute. A gel-filtration standard (Bio-Rad Laboratories, Hercules, CA) was used for determination of the molecular weight. All procedures were conducted at 4°C.
LO Hydrolysis Assay in Chromatography Fractions.
For assay of LO-hydrolyzing activity in the gel-filtration chromatographic fractions, an aliquot of sample was incubated at 37°C for 1 hour with 420 μM (200 μg/ml) LO. The reaction was terminated by mixing with an equal volume of acetonitrile. The sample was added an aliquot of IS and then filtered. The resulting sample was injected in the LC-MS system consisting of 1200 LC/MSD coupled to 1200SL (Agilent Technologies, Santa Clara, CA). The analyte was separated on an analytical column, an Asahipak NH2P-50 2D (5 μm, 2.0 × 150 mm; Showa Denko K.K., Tokyo, Japan), with an isocratic mobile phase consisting of 10 mM ammonium acetate/acetonitrile (45:55). The flow rate employed was 0.3 ml/min. The analyte was detected with the electrospray ionization (ESI) source operated in positive-ion mode. MS data were acquired in selected ion monitoring mode at m/z 347 and m/z 350 for laninamivir and IS, respectively. For APT1 inhibitory assay, the reaction mixture was incubated with 420 μM LO in the presence of 0.1 μM palmostatin B. One unit of LO-hydrolyzing activity was defined as the activity required for 1 nmol/ml production of laninamivir.
Protein Identification and Quantification in Chromatographic Fractions.
Two hundred fmol of bovine serum albumin (BSA) was added as IS to an aliquot of each fraction from gel-filtration chromatography. Then, each fraction was subjected to methanol/chloroform precipitation, in-solution reduction and alkylation, followed by trypsin digestion, in a similar manner with our previous report (Sakurai et al., 2013). In brief, proteins were precipitated with methanol/chloroform precipitation (Wessel and Flügge 1984) to remove salts and detergents, then reduced with dithiothreitol followed by alkylation with iodoacetamide, and digested with trypsin. Resulting peptides were desalted with StageTips (Rappsilber et al., 2007) and dissolved with 12 μL of 5% formic acid. An aliquot of the peptide mixtures (4 μL) was injected into the LC-MS/MS system consisting of LTQ-Orbitrap ELITE coupled to Easy-nLC II (Thermo Fisher Scientific, Waltham, MA). The peptides were eluted from the in-house packed tip column (140 mm × 75 μm I.D., 3 μm Inertsil C18; GL Sciences, Tokyo, Japan) with a linear gradient of acetonitrile in 0.1% formic acid at a flow rate of 200 nL/min. Protein identification and quantification were performed by the MaxQuant (version 1.2.0.5; Max Planck Institute of Biochemistry, Martinsried, Germany) (Cox et al., 2009, 2011). Briefly, MS/MS spectra were searched against UniprotKB/Swiss-Prot human data downloaded on November 28, 2012. The identified proteins were quantified in an ion intensity-based label-free algorithm implemented in MaxQuant, which was a relative quantitation algorithm using the ion intensity of identified peptides in LC-MS without stable isotope labeling technology such as SILAC (Ong et al., 2002), iTRAQ (Ross et al., 2004) or AQUA (Gerber et al., 2003). Two missed cleavages were allowed, along with carbamidomethylation of cysteine as a fixed modification; variable modifications were oxidation of methionine and acetylation of N-term amino acid of the protein. Mass tolerance for precursor ions was 7 ppm, mass tolerance for fragment ions was 0.5 Da, and false-discovery rates at peptide and protein levels were less than 0.01. The proteins identified by more than one single peptide were used for further correlation analysis.
After MaxQuant analysis, all proteins quantified in each fraction were normalized by the amount of BSA to account for any experimental error and to correct any variations in sample complexity. Pearson correlation coefficient was calculated between the normalized abundance profile for each protein and LO-hydrolyzing activity profile.
The total protein concentration was determined by modified Bradford protein assay (Coomassie Plus Protein Assay, Thermo Fisher Scientific) using BSA as a standard protein. Chromatographic fractions were assayed before a spike-in of BSA as IS for label-free quantitation.
The immunohistochemical assay was performed on formalin-fixed, paraffin-embedded human pulmonary tissue sections using Dako Autostainer Link 48 (Dako Denmark A/S, Glostrup, Denmark). Deparaffinization and target retrieval were processed on the tissue sections in a single step using Dako PT Link (EnVision FLEX TRS Low pH), where the optimal incubation was set at 97°C for 20 minutes. Endogenous peroxidase activity was quenched with peroxidase-blocking solution (Dako) for 5 minutes, and nonspecific antibody binding was blocked with protein block, serum free (Dako) for 30 minutes. Then, anti-ESD antibody or anti-APT1 antibody (2 μg/ml) was applied as a primary antibody on the sections and incubated for 45 minutes, and isotype antibody (2 μg/ml) was used as a negative control. EnVision+System-HRP Labeled Polymer Anti-Rabbit was reacted as a secondary antibody for 30 minutes, and antibody complex was visualized after the addition of 3,3′-diaminobenzidine tetrahydrochloride (DAB)+, liquid (5 minutes × 2; Dako). The section was counterstained with hematoxylin.
Enzymatic LO Hydrolysis in Human Pulmonary Subcellular Fractions.
LO-hydrolyzing activity in human pulmonary S9, cytosol, and microsomes is shown in Fig. 2A. At 10 μM LO, activity was observed in all of the subcellular fractions tested, with almost the same levels per milligram protein basis (1.2–1.9 pmol/min/mg). Furthermore, for the inhibitory effect of typical esterase inhibitors, LO-hydrolyzing activity in pulmonary S9 was strongly inhibited by a potent serine esterase inhibitor DFP at 0.1 mM or more. DTNB, known as an inhibitor of thiol-containing esterase, exhibited weaker inhibitory effects than DFP, whereas much weaker inhibition was observed in the treatment with BNPP (carboxylesterase inhibitor). Eserine (cholinesterase inhibitor) showed no enzymatic inhibition (Fig. 2B).
Enzymatic LO hydrolysis in human pulmonary subcellular fractions. (A) LO-hydrolyzing activity in human pulmonary subcellular fractions (S9, cytosol, and microsomes). Each bar represents the mean ± S.E. in triplicate determinations. (B) The inhibitory effect of typical esterase inhibitors on LO-hydrolyzing activity in human pulmonary S9. Each bar represents the mean of triplicate determinations.
Selection of Possible Candidate Proteins for LO-Hydrolyzing Enzymes.
A schematic diagram for this study is illustrated in Fig. 3. As shown in Fig. 2A, LO-hydrolyzing activity in pulmonary S9 was substantially derived not only from the soluble fraction (cytosol) but also from the insoluble fraction (microsomes). Therefore, nonionic detergent NP-40 was added to the S9 fraction to solubilize membrane proteins without altering biologic activity, and the centrifuged soluble fraction (overall recovery: 73.8%) was subjected to a single-step gel-filtration chromatography. From the enzyme assay for the chromatographic fractions collected, LO-hydrolyzing activity was separated into two distinct peaks (Fig. 4), which were designated as peak I (fraction no. 26-32) and peak II (fraction no. 33-39), respectively. The contribution rate to total activity was estimated to be approximately 40% and 30% for peak I and peak II, respectively, based on the calculation of each peak area (%) of LO-hydrolyzing activity in the gel-filtration fractions. Then, to seek proteins that showed a high correlation with LO-hydrolyzing activity, all proteins in the fractions were identified and quantified by LC-MS/MS analysis.
A schematic diagram of proteomic correlation profiling to identify the candidates for LO-hydrolyzing enzymes.
Selection of possible candidate proteins demonstrating LO-hydrolyzing activity in human pulmonary tissue. After fractionation of 105,000g supernatant of NP-40 treated human pulmonary S9 by gel-filtration chromatography, an aliquot of each fraction was assayed for the LO-hydrolyzing activity (solid line, left y-axis) and protein concentration (open bar, right y-axis). The positions of the native molecular weight markers are indicated at the bottom.
Theoretically, protein of a higher correlation coefficient has higher probability of a responsible protein to the activity profile, and our previous experiences (Kubota et al., 2009; Sakurai et al., 2013, and unpublished results) are in agreement with this concept. In total, 2381 proteins were identified, but there was no single protein that had the bimodal chromatographic peaks highly correlated with the enzyme activity profile (data not shown). Therefore, it was speculated that the active peaks consisted of two individual enzymes, which were independently derived from peak I and peak II. Possible candidate proteins were listed for each peak based on the correlation coefficient with the enzyme-activity profile. With respect to peak I, there were 1160 proteins identified, and the top 30 most correlated proteins were listed in Table 1. In a similar manner, with regard to peak II, 1003 proteins were identified, and the top 30 proteins were also placed in descending order of correlation coefficient, as shown in Table 2. Among the proteins that were annotated as "hydrolase," ESD (R2 = 0.9432) and APT1 (R2 = 0.9765) were finally selected as the most possible candidates for the peak I- and peak II-derived proteins, respectively. ESD and APT1 proteins were clearly detected in the peak I and peak II fractions, respectively, by Western blot analysis (data not shown).
Among the 1160 proteins identified, the top 30 candidates with high correlation coefficient between relative intensity of the identified proteins and LO-hydrolyzing active peak I (fraction no. 26-32) on gel-filtration chromatogram are listed. Identified peptides for each protein are listed in Supplemental Table 1.
Among the 1003 proteins identified, the top 30 candidates with high correlation coefficient between relative intensity of the identified proteins and LO-hydrolyzing active peak II (fraction no. 33-39) on gel-filtration chromatogram are listed. Identified peptides for each protein are listed in Supplemental Table 2.
Identification of ESD and APT1 as LO-Hydrolyzing Enzymes.
To investigate whether both ESD and APT1 are authentic candidates for the LO-hydrolyzing enzymes, their recombinant proteins (rESD and rAPT1) were subjected to the determination of LO-hydrolyzing activity. As shown in Table 3, the enzyme activities in rESD and rAPT1 were 232 ± 16 and 377 ± 36 pmol/min/mg at 10 μM LO, respectively, both of which were more than 100-fold higher than that in human pulmonary S9 (1.56 ± 0.05 pmol/min/mg). On the other hand, BSA (negative control) had a negligible activity.
Candidate protein recombinants (rESD and rAPT1), pulmonary S9, and BSA (negative control) were incubated with 10 μM LO, and the enzyme activity was tested. Each value represents the mean ± S.E. in triplicate determinations.
Inhibitory Effect of Palmostatin B on Chromatographic LO-Hydrolyzing Activity.
Palmostatin B is known as a specific APT1 inhibitor (Dekker et al., 2010, Rusch et al., 2011), although no specific inhibitor has been reported for ESD. LO-hydrolyzing activity profiles were measured for the fractions separated by gel-filtration chromatography, in the absence or presence of a specific APT1 inhibitor, palmostatin B (Fig. 5). In the absence of palmostatin B, there were two active peaks, peak I and peak II, on the chromatogram. On the other hand, in the presence of palmostatin B, peak II-derived activity was mostly inhibited without any significant effect in peak I–derived activity, supporting APT1 as a major enzyme in peak II.
Inhibitory effect of palmostatin B on LO-hydrolyzing activity in the fractions eluted from gel-filtration chromatography. An aliquot of each fraction was assayed for LO-hydrolyzing activity in the absence and presence of a specific APT1 inhibitor, palmostatin B (0.1 μM). The positions of the native molecular weight markers are indicated at the bottom.
Immunohistochemical Localization of ESD and APT1 in Human Pulmonary Tissue.
Figure 6 shows representative immunohistochemical images of ESD and APT1 in human pulmonary tissue sections stained with anti-ESD antibody (A) and anti-APT1 antibody (B), respectively, using isotype antibody (C) as a negative control. As shown in Fig. 6A, ESD was highly expressed in the pulmonary epithelia with a strong dot-like or granular stain being localized in the cellular cytoplasm on the side of the luminal membrane. No obvious staining of ESD was observed in other tissue components. In Fig. 6B, APT1 was also highly expressed in the pulmonary epithelia, with overall cellular staining including the cellular cytoplasm. Positive staining of APT1 was observed in the alveolar macrophages as well. In the case of the isotype antibody (Fig. 6C), there was little or no cross-reactivity with cell surface antigens on the tissue section.
Representative immunohistochemical images of ESD and APT1 in human pulmonary tissue. Human pulmonary tissue sections were stained with anti-ESD antibody (A), anti-APT1 antibody (B), and isotype control (C). After treatment with horseradish peroxidase–labeled anti-rabbit IgG, the antibody complex was visualized as a dark-brown precipitate by using DAB (3,3′-diaminobenzidine tetrahydrochloride) as a substrate.
In this study, we have successfully identified two hydrolyzing enzymes, ESD and APT1, which would be primarily involved in the bioactivation of the anti-influenza prodrug LO in human pulmonary tissue, using a versatile methodology named proteomic correlation profiling (Kubota et al., 2009; McAllister and Gygi 2013; Sakurai et al., 2013). The proteomic correlation profiling has a feature to facilitate rapid identification of the responsible protein from crude complex lysate with fewer purification steps using a smaller starting material, as compared with the conventional methodology. Actually, in single-step gel-filtration chromatography using only approximately 2.5 mg of the human pulmonary S9 fraction, we could identify two highly possible candidates of LO hydrolases, ESD for peak I and APT1 for peak II (Fig. 4).
ESD (EC 3.1.2.12), also known as S-formylglutathione hydrolase and methylumbelliferyl-acetate deacetylase, is a serine hydrolase that belongs to the esterase D family. This protein is expressed in the majority of normal tissues and is localized in the cytoplasm and cytoplasmic vesicles. ESD catalyzes the hydrolysis of S-formylglutathione, an intermediate of the glutathione-dependent pathway of formaldehyde detoxification, converting to the reduced forms of formic acid and glutathione (Eiberg and Mohr, 1986; Harms et al., 1996). In addition, ESD can act as a carboxylesterase and hydrolyze a variety of ester substrates including O-acetylated sialic acids. For example, it has been reported that ESD can specifically cleave the O-acetyl ester linkage at the exocyclic C9 position of 9-O-acetyl-N-acetylneuraminic acid (Neu5, 9Ac2), suggesting that it is involved in the recycling of sialic acids (Varki et al., 1986). From a chemical structural point of view, LO has an O-octanoyl ester linkage on its sialic acid-like structure that is very similar to that of Neu5, 9Ac2. This structural feature supports the reasonability of LO hydrolysis by ESD. In our experiment, recombinant ESD had distinct LO-hydrolyzing activity (Table 3), suggesting a responsible enzyme for LO hydrolysis. In addition, it was consistent with ESD as one of the LO hydrolases that the LO-hydrolyzing activity in pulmonary S9 was strongly inhibited by DFP, a serine hydrolase inhibitor (Fig. 2).
Similarly to ESD, sialate O-acetylesterase (SIAE) is known as an enzyme-hydrolyzing O-acetyl ester linkage of Neu5, 9Ac2 (Schauer 2000; Angata and Varki, 2002). Moreover, ESD has been reported to show significant carboxylesterase activity against the following model substrates: α-naphthyl acetate and p-nitrophenyl acetate (Degrassi et al., 1999). A partial contribution of carboxylesterase on the LO hydrolysis was also supposed from the data that carboxylesterase inhibitor BNPP showed a weak inhibition against LO-hydrolyzing activity with 42.3% inhibition at the highest concentration (1 mM; Fig. 2). Considering these situations, we additionally investigated the potential of LO-hydrolyzing activities of SIAE and CES1 (liver carboxylesterase 1), using their recombinant proteins. However, there was little or no LO-hydrolyzing activity in either protein (data not shown), indicating that these two enzymes were not responsible for the LO bioactivation although they were identified as candidate proteins for peak I and placed in a higher rank than ESD (Table 1). No LO-hydrolyzing activity was clearly observed in LTA4H (leukotriene A-4 hydrolase) as well, using its recombinant protein (data not shown). Nevertheless, the involvement of other enzyme(s) cannot be completely ruled out because some proteins with hydrolase annotation still remain as candidate proteins for peak I, such as PGAM1 (phosphoglycerate mutase 1), RCL (deoxyribonucleoside 5-monophosphate N-glycosidase), DDX39B (spliceosome RNA helicase DDX39B), and IAH1 (isoamyl acetate-hydrolyzing esterase 1 homolog). However, from enzyme activity using recombinant ESD (Table 3), it is highly plausible that ESD is at least one of the component enzymes consisting of LO-hydrolyzing activity for peak I.
APT1 (EC 3.1.2.-), also known as lysophospholipase 1 (LYPLA1), is a serine hydrolase that belongs to the α/β hydrolase superfamily. This protein is expressed in various tissues including the gastrointestinal tract, respiratory epithelia, salivary glands, and liver, and is localized predominantly in the cytoplasm (Sugimoto et al., 1996; Wang et al., 1999; Hirano et al., 2009). APT1 works as a thioesterase that catalyzes depalmitoylation of H-Ras (Duncan and Gilman, 1998), G protein α subunit (Duncan and Gilman, 2002), and the endothelial isoform of nitric oxide synthase (Yeh et al., 1999). Furthermore, APT1 was also reported as an oxyesterase that cleaves the octanoyl group of ghrelin (Shanado et al., 2004; Satou et al., 2010). From the similarity of chemical structure with ghrelin, APT1 was hypothesized to be the most promising candidate for peak II, and this hypothesis was supported by LO-hydrolyzing activity of recombinant APT1 (Table 3). Moreover, as shown in Fig. 5, LO-hydrolyzing activity of peak II was almost inhibited by palmostatin B, a specific APT1 inhibitor (Dekker et al., 2010; Rusch et al., 2011). Also, in the same way as ESD, a strong enzyme inhibition by DFP (Fig. 2) was consistent with the fact that APT1 is categorized in the serine hydrolase class of enzymes. These results demonstrated that APT1 consisted mostly of active peak II.
Immunohistochemical analysis showed that ESD was highly localized in the cytoplasm and/or cytoplasmic vesicles of pulmonary epithelia (Fig. 6A), whereas APT1 was highly expressed in the overall pulmonary epithelia and alveolar macrophages (Fig. 6B). According to our previous reports, active metabolite laninamivir was highly maintained in the ELF and alveolar macrophages after inhaled administration of LO in healthy volunteers (Ishizuka et al., 2012). In addition, in mice intranasally administered LO, the laninamivir was also deposited in the pulmonary tissue, especially in the epithelia. These results indicated that LO could be hydrolyzed into laninamivir in the pulmonary epithelia and/or alveolar macrophages, in which ESD and APT1 were mainly localized.
Prodrugs require bioactivation by the metabolizing enzymes to demonstrate its therapeutic effect. Genetic polymorphism in the prodrug-activating enzymes can cause a crucial impact on the interindividual variability in the drug exposure and response, as reported in some drugs, such as clopidogrel (Shuldiner et al., 2009), codeine (VanderVaart et al., 2011), and tramadol (Gan et al., 2007). Regarding the LO-hydrolyzing enzymes, the genetic polymorphism has been identified for ESD in a previous report (Ebeli-Struijk et al., 1976), in which the ESD*2 allele was commonly observed with a different gene frequency depending on ethnic group. Additionally, it has been reported that the enzyme activity associated with ESD*2 was estimated to be 40% lower than that associated with ESD*1, using a typical substrate 4-methylumbelliferyl acetate (Horai and Matsunaga 1984). However, in the case of LO, this genetic factor would be unlikely to make a huge influence on the interindividual variability because at least the two enzymes (ESD and APT1) were involved in the LO bioactivation and the contribution of multiple enzymes in the same reaction is considered to dilute the potential impact on the interindividual variability. Further assessment on this issue should be performed more precisely in the future.
In conclusion, this study demonstrated that both ESD and APT1 would be key enzymes responsible for the bioactivation of LO in the human pulmonary tissue. These enzymes could play an important role for exhibiting anti-influenza virus activity when LO reached the respiratory tract after inhaled administration in humans.
The authors thank Hidetaka Sakurai and Hiroshi Katsuno for experimental suggestions and contributions to this work. The authors also thank Dr. Makoto Yamashita for many helpful discussions.
Participated in research design: Koyama, Ogura, Nakai, Kubota.
Conducted experiments: Koyama, Ogura, Watanabe, Oofune.
Contributed new reagents or analytic tools: Koyama, Ogura.
Performed data analysis: Koyama, Ogura, Munemasa.
Wrote or contributed to the writing of the manuscript: Koyama, Ogura, Nakai, Kubota, Shinagawa, Izumi.
K.Ko. and Y.O. contributed equally to this work.
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Tv switches on then goes off after 5 secs.Have changed power board, Will stay on if holding the on button in , but once released goes off. any ideas.I have also changed the switch card to no avail.
I don't know much about TV's, but to me it sounds like the problem might be related to your power button.
Sharp Aquos lc-70le640u Blank screen, flashing power light.
I have my sony dvd player hooked up to my sharp roku tv I can hear the sound but no picture?


Part 2: Get Ready for Outdoor Living!
For us, a big part of any successful outdoor room are the soft goods – the fabrics used on the pillows, drapes and accessories throughout this unique space. Because it's situated outdoors, it's open to the elements and all that brings with it. Although it's nice to think you'd bring your cushions and pillows inside whenever they're not in use, that's not always realistic. And, we aim to create spaces clients love to live in and with, not create more work for them.
Outdoor living – perfect for the whole family!
So, we turn to Sunbrella fabrics, the ultimate in textiles for outdoor living. You may have heard of Sunbrella before, probably in terms of awning solutions for outdoor spaces. And while these can be nice, for the right home, there's so much more to Sunbrella now!
Ottomans and poufs! This ottoman in a textile design by our friends at Laurie Bell, another high performance fabric line offered by steve mckenzie's, offers such versatility! Extra seating, a place to lounge your feet or place a tray of beverages – every outdoor space should include an outdoor ottoman or pouf.
Throws. The ultimate in outdoor comfort and ideal for those chilly summer nights. Toss a few outdoor throws throughout your space and you've instantly made it more inviting!
And this one may be our favorite – dog beds! We couldn't imagine a better use for outdoor fabrics than for dog beds. You'll need something extremely durable, and it doesn't get better than this. And these stylish beds, thanks to Laurie Bell, flow seamlessly with our decor. Sometimes we think the steve mckenzie's shop dogs, Bisi and Duke have it made!
If you're really looking to create an oasis, outdoor drapes are where it's at. They dance in the breeze and have the ability to turn just about any space into a tropical paradise. With the color, pattern and texture options now available, we're only limited to our imaginations!
Phew! After all that, we think it's time for a sit and glass of iced tea!
If you'd like additional details about Sunbrella fabrics and how the steve mckenzie's team can create your idea of a fabulous outdoor space, feel free to reach out to us and learn more. We look forward to working with you and creating an amazing outdoor room. Soon you'll have it made in the shade!
Previous Happy Staint Patrick's Day from steve mckenzie's!


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Home » News & Events
Associate Director presents at the first DHS Science and Technology(S&T) Research Council Webinar
ALERT Associate Director, Carey Rappaport was invited to speak at the first DHS Science and Technology(S&T) Research Council Webinar on Monday, October 22nd. Prof. Rappaport participated in the Explosives Detection Panel, which discussed the Quadrennial Homeland Security Review mission flow down to potential technology solutions supported by S&T with subject matter experts (SMEs) from DHS labs, DOE National Labs, and S&T Centers of Excellence.
In addition to Prof. Rappaport, the panel also included:
Jon L. Maienschein, Ph.D.
Director, National Explosives Engineering Sciences Security Center
Director, LLNL Energetic Materials Center
Leader, PLS Energetic Materials Group
Physical and Life Sciences Directorate
Richard T. Lareau, Ph.D., Chief Scientist,
U.S. Dept. of Homeland Security
Science and Technology Directorate
Transportation Security Laboratory
David Atkinson, Ph.D., Chief Scientist
Explosives Detection Research
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redpajama_set_name: RedPajamaCommonCrawl


As planning applications go, it would be fair to say that case #10/90250/FULODA, submitted to the London boroughs of Newham and Waltham Forest planning committees in May 2010, stood out somewhat. In among the loft conversions and Victorian conservatories that mark the staple fare of the weekly planning agenda in this part of east London, this particular file put the sober case for a 115m steel tower in the form of a vast, deconstructed spiral, painted bright red, lit up at night and visible from 10km away. Did the neighbours mind?
By the time it reached the application stage, the creators of the ArcelorMittal Orbit on the Olympic site (or "Boris's Folly", as it was generally known on the blog sites) had already invited as many neighbours as possible to comment. The Big Opportunity, a conglomeration of interest groups in the vicinity, with 56 members ranging from the East London Inventors Club to the Ladies' Wing of the Followers of His [Hindu] Holiness Swaminarayan Mandir, had been consulted. Responses had been invited from interested individuals from the Orbit's "region", which stretched as far as Milton Keynes, Brighton, Canterbury and Southampton. From all this reaching out, 118 comments had been received and noted by the time of the full planning application: 39% wrote in favour of a design variously described as "beautiful", "fragile" and "feminine". The rest argued in forceful terms that it was "ugly" and "not symmetrical" and objected in no particular order to the fact that it was red, pointless, expensive and an advert for Arcelor Mittal (and quite a cheap one at that).
At an open planning meeting, one of the tower's creators, the engineer Cecil Balmond, who is responsible for some of the world's most inspired and innovative structures, recalls how he thought they had lost it. "From the floor, people just seemed to be lining up with complaints, one after the other," he recalls. "It looked pretty bad at one point. We don't want this and what is the point of that? But then after a while came the counter-arguments: Britain needs something different and new, we can't bury our heads in the sand, all that. I just stood back and listened."
By the time of that public debate, Balmond and his fellow Orbit-creator, the artist Anish Kapoor, had become rather used to explaining their ideas to committees and taking feedback. They had (mostly calmly) addressed the concerns of critics, conservationists, health and safety officers and legacy deliverers one by one. Rather than calling it a tower, they liked to refer to the Orbit as "the tallest sculpture in the UK". In response to a suggestion that this sculpture had no relevance for London or the Olympics, it was argued that "the Orbit will take on a relevance of its own" after the Games had ended. As detractors had correctly observed, the colour red was chosen "intentionally for it not to blend with its surroundings". Charged with asymmetry, they argued that it was "meant to look unstable or fluid". Those who were standing up for the beleaguered bat colonies in the area had little cause for concern either: the low levels of light on the Orbit "would have no discernible effect on the bat assemblage over the Olympic site" or, indeed, on human assemblages in the neighbouring streets.
Last week, in advance of the tower's opening, I went to talk to Balmond and Kapoor at their respective studios about how they managed to stay sane and see this strange project through. In a way, they are typical Londoners. Balmond was born in Kandy, Sri Lanka, Kapoor in Mumbai, India. They both came to England as students and never left. Balmond has his hi-tech base, all 3D printers and biomorphic structures, on the edge of Hackney, a mile or two from the Olympic park; Kapoor's studio is a linked complex of factory spaces that stretches all the way down a road in Camberwell, south of the river (as his fame and ambition have spread so has his workshop; it now has the feel of a kind of aerospace lab manned by medieval guildsmen). In each man's office, scale models of the Orbit have pride of place. And despite what has been a gruelling process, both Kapoor and Balmond retain a sense of boyish excitement – or perhaps simple relief.
Kapoor started out in his teens with ambitions to be an engineer and this project has more than satisfied any remaining vestiges of those dreams: "I hope," he says, "I always will have a fascination with that archaic, elemental need to feel like an ant in an ant colony. To climb up the pyramids and just feel awe at man-made structures. That was the attraction of this for me."
For a role model in that enthusiasm, Kapoor needed to look no further than the project's driving force. Boris Johnson was almost lost for superlatives when announcing that work was starting on his great scarlet tower in 2010: "It would have boggled the minds of the Romans," the mayor declaimed. "It would have dwarfed the aspirations of Gustave Eiffel, and it will certainly be worthy of the best show on Earth, in the greatest city on Earth."
That was certainly the idea to begin with. The story goes that Johnson, keen to make his mark on the Olympic site that had become the fiefdom of the Tory peer Lord Coe, bumped into Britain's richest man, Lakshmi Mittal in the lavatories at the World Economic Forum in Davos in 2009. Grasping his opportunity with both hands, the mayor buttonholed the steel magnate about the possibility of funding a lasting symbol of London 2012, boggling the minds of Romans etc. Mittal himself confirms to me that "Boris might have even taken less time than he says to convince me... sometimes you just hear an idea that resonates with you - this was one of them." Soon thereafter, Mittal pledged £17m of his fortune to Boris's priapic fantasy and the mayor sent out a notice inviting the artists and architects of his realm to find a way of spending that money.
"Anish called me up that morning," Balmond recalls. The pair had long been friends and had collaborated on various projects including Kapoor's Marsyas, the brilliant crimson horn that filled the Turbine Hall at Tate Modern in 2002. "He said, 'Have you seen this one?' I hadn't. Then he said, 'Shall we get together and do this? You know, rival the Eiffel Tower and so on?' And I thought, 'Well, no one's going to say no to that.' So we joined up. And then realised that there wasn't the money for the Eiffel Tower."
Kapoor and Balmond sat down with a sketch pad and thought what the reference points might be. As well as Eiffel, they thought of Tatlin's Tower (the vast constructivist monument conceived for Petrograd in the year of the Russian Revolution, but never built). And they thought, too, of the Tower of Babel, particularly Bruegel's version of it, an irregular mass of stone and humanity reaching chaotically heavenwards, like some termite's mound. And then they thought: how can we make a mythical tower new?
"Anish was saying, 'Well, all towers go up, but what can we do that is different?'" Balmond recalls. He couldn't imagine to start with. "But then I thought, everything that goes up is concentric, essentially. That's what we need to get away from. So I thought 'orbit', just as a metaphor originally." He sketched a loose ellipse on a piece of paper. "Now planetary orbits are highly unstable things, whirling around, but they are stable in the sense that they follow a fixed path. So then I drew an orbit that comes back on itself but keeps touching itself. So that was the idea."
It was to be 180m high, the platforms just stuck in as and where. With this sketch, from a starting line-up of 60 proposals, Balmond and Kapoor made the last three, alongside Antony Gormley, looking to recreate the success of his Angel of the North, and the Hackney-based architects Caruso St John. Just before he walked in to present to the first of many committees, Balmond recalls: "Someone from the mayor's office said to me, 'Do you know the budget?' And I didn't. She said it was around £25m. And I thought, 'Oh Christ!' Because what we had I knew would cost £50m to £80m. So straight away we brought it down to as low as we could go and still get a good sightline into the stadium: 115 metres."
That was only the first of a series of compromises. In this sense, as Kapoor observes the Orbit is very much of its political moment: "The basic premise was to do everything you promised for about half the money," he says, with a grin. Earlier in the week he had watched the Olympic mockumentary Twenty Twelve's take on the process. "The organising committee on the show come up with the idea that Orbit should be a symbol for promoting sexual health," he says. "But sadly they copped out half way through and don't end up putting a condom on it as planned. What is astonishing about it is how accurate it was in terms of some of the meetings we all had..."
As Balmond says, with a similar sense of weary mischief: "I suppose the story behind the story is that the competition seemed to go on for ever, round after round." The decision process lasted the best part of a year. "At one of these meetings, I said to Boris, 'Just choose someone, for God's sake. Otherwise nothing will get built.'"
Balmond and Kapoor not only had to convince Nicholas Serota and his aesthetic jury of the value of the design, but also the "legacy committee", who, full of Dome-shaped nightmares, didn't want a "white elephant, still less a red one". So there was insistence on maximum retail and restaurant areas. The elevator had risen up the outside of the tower in the original plan but that cost too much so they put it inside one of the legs. The walkways that spiral up to the viewing areas were originally open but health and safety insisted they be covered. Gaps between stair treads were also removed. "First, any space had to be too narrow for a mobile phone," Balmond recalls, "then it was a 50p piece."
After that, the Olympic delivery people, who were building the stadium site, "were instinctively against it because they had done a brilliant job of getting things ready on time and they didn't necessarily want this huge art piece in the middle of it all, potentially screwing all their plans up".
In order to minimise disruption, the Orbit was put up without scaffolding, and essentially by three men: one in a crane and two rising slowly on cherry pickers, bolting the ultimate Meccano together piece by piece. And, despite all the earlier compromises, both Balmond and Kapoor are immensely satisfied with the result, though they are tired of the question: "What is it?"
"The fact is that you will never get Orbit in 2D," Balmond says. "Its richness and its over-layers can look excessive to a certain kind of mind. But 3D and the scale are the only way to judge the piece. Even then, it's a tough aesthetic for some."
"The problem with models," Kapoor says, "is that you can't pretend scale. You have to experience it."
With this in mind, early on the morning after I had spoken to Balmond and Kapoor, I drove east to have a look at their creation. As I came down from the A12 flyover, the Orbit was rising into the gloomiest morning, like some strange helter-skelter, defiantly red against the black storm clouds (Boris Johnson's greatest regret is that it did not incorporate a slide to whizz down). The Olympic development has sought to make sense of the particularly chaotic bit of urban landscape that the tower presides over; it hasn't succeeded quite and the tangle of the Orbit seems, from all the vantage points I could find, to add to the confusion. The closer you get to it, the less sense you can make of it, beyond a smile-inducing kind of energy and movement. Which is, for better or worse, exactly what Kapoor and Balmond (and perhaps Boris and Mittal) had in mind.
You can see in it what you want, as Balmond observes. Mittal tells me that to him the Orbit "represents the essence of what the Olympics are about, pushing yourself to the limit... building the unbuildable..." (though he also likes the fact that the structure that bears his name is "a showcase for everything steel has to offer...") Pandering a little to his sponsors Balmond admits he did one "cheeky presentation" where he extrapolated the five Olympic rings from the swirl, "a bit of post-rationalisation, but they are there". More than that, though, he claims to see "a kind of semi-organised flux, which was a pretty good way of describing London in the 21st century, and all its energy frothing and bubbling round and around." That kind of thing.
Kapoor's worst nightmare, he said, would have been to create a logo or, worse, a national symbol in the manner of the Beijing Olympics. "I can clearly make sleek objects but this was not meant to be one of them." So what was it meant to be?
"It's a series of discrete events tied together," Kapoor says, which again is something approximating his idea of London. "We didn't want an icon, we wanted a kind of moving narrative. You start under this great domed canopy that sits above you, almost ominous darkness, sucking you in. Then you come up slowly to light. At the top, there is a room with two very large concave mirrors, bringing the sky in, as if you are in the lens room of a telescope. There are moments, walking round, when it looks a jumbled mess, and then at certain points you might see little harmonies and clarity. That is the kind of thing we wanted, not something that gave itself away all at once."
Kapoor and Balmond can talk about their creation in this way because they have had to. But they prefer simpler notions really. "It was just an attempt to answer the question: how do you go up if not in straight lines?" says Balmond, who plans to watch the 100m final from the top. Kapoor, meanwhile, sums up his sense of his creation with a final laugh. "Don't you think it's just amazing that they actually let us build this?" he asks, with undimmed incredulity. And the more you look at it, the more you agree.


Powered by Flick Anticimex, our Amalgamated Pest Control branch in Muswellbrook provides pest control and termite inspections services.
Flick Anticimex has merged with Amalgamated Pest Control (APC) and the existing APC offices now operate as one Flick Anticimex team! In September 2016, APC and Flick joined forces to become the largest pest control company in Australia. Located in Denman, our joined Flick Anticimex Newcastle and APC office are a trusted and reliable team, made up of Muswellbrook locals. As trusted household names, our local experts conduct safe and effective pest control services for homes and businesses in Muswellbrook. Servicing Australian homes since 1918, our team of technicians know your area and know the local pest problems that plague your community. Give our Muswellbrook office a call on 02 4911 7200, where we can arrange a pest inspection and quote you a competitive price.
Termites (also known as white ants) are terrors to households across Australia. With 1 in 3 homes being ravaged by termites, Muswellbrook residents need to take extra precaution when it comes to termites. Early detection and preventative inspections carried out on the property can reduce the cost of repairs. The most effective method of protection against termites is by conducting annual termite inspections on your home or business, depending on the amount of wood used in the home and other factors. Some homes should even consider a termite inspection anywhere between 3 to 12 months. Following an inspection, the technician will provide a detailed treatment or prevention program and quotation; which may include recommendations for monitoring, baiting systems, chemical soil treatments and termite colony location and eradication. Termite colonies can be found hidden in timber wall frames and roofs. They eat the timber frames and any wood or cellulose found in and around your house. Flick and APC can perform a thorough termite inspection and then we will perform a liquid perimeter treatment. Regular check-ups are recommended to ensure that your property is safe from severe structural damage. Termite control should also be considered before you purchase your home. Flick Anticimex and APC can conduct pre-construction or pre-purchase pest inspections. A simple termite inspection by one of our professionals could save you thousands of dollars in repairs.
Possums are a beautiful part of Australia's native wildlife, but they are able to pose a threat to your property and to themselves. Their urine and droppings can negatively affect your health. They also expose electrical wires, and can often become stuck within the roof cavity of your home. These animals are protected by the Nature Conservation Act 1992. Harming them in any way is forbidden, as is keeping them without permission from the Department of Natural Resources and Environment. Break these rules and you could find yourself slapped with a very heavy fine. A licensed pest manager from APC and Flick Anticimex can make a house call and set a trap on your roof. Before you know it, the possum will have been caught, temporarily relocated, and then set free again. The possum will be returned to your area, but we will provide homes for the possums, so they don't have to find shelter in your home.
Muswellbrook's rich array of gourmet food and wine produce in addition to the local creek creates an ideal breeding ground for rodents. These rodents are often found hiding out in roofs and wall cavities. They can cause serious damage to property caused by gnawing everything in their search for nesting sites and food. Rats and their droppings are also known to spread disease. To make matters worse, if you live in the vicinity of restaurants or similar businesses, the risk of rat infestations is greatly increased. Our Muswellbrook pest control team rids properties and homes of rats and mice. We also provide commercial rodent control solutions that can help restaurants and similar businesses solve their rodent problems.
Amalgamated Pest Control and Flick Anticimex serve households and businesses in Muswellbrook and all surrounding suburbs. Find out if your suburb falls under our service area by calling our team today. Call us at 02 4911 7200!


text:
Q: [console]::KeyAvailable is not identified while running from startup The script task is to respond to keyboard commands 'F3' and 'F4'
When running the script from terminal or CMD it is fully functional
However, when calling the script from start-up cmd, the script is running but not receiving the commands from keyboard
Any idea why behavior is different when running from startup ?
This is the command line:
powershell  -WindowStyle hidden -ExecutionPolicy Bypass C:\PowerShell\KeyBoardTest\Keyboard-Command-Response.ps1 >>  C:\PowerShell\KeyBoardTest\Keyboard-Command-Response_log.log

This is the PowerShell script:
$shell = New-Object -ComObject "Shell.Application"
$Pause_script = 0
$exit_command = 'FALSE'

Sleep -Seconds 3
$Res= $shell.minimizeAll()

While ($exit_command -eq 'FALSE')
{
    Sleep -Seconds 1
    Write-Output "Mylog: Waiting to Keyboard"
    if ([Console]::KeyAvailable)
    {

        $KeyPressed = [System.Console]::ReadKey()
        Write-Output "Mylog: Identified Press of key"
        switch ($KeyPressed.key)
        {
        F3 {
            if ($Pause_script -eq 0){
                $Pause_script = 1
                Write-Output "Mylog: F3 Detected, Pausing Run"
                }
            else{
                $Pause_script = 0
                Write-Output "Mylog: F3 Detected, Resuming Run"
                }
            }
        F4 {
            $exit_time = Get-Date
            [DateTime]$Date = $exit_time
            $mytime = $date.ToShortDateString() + " " + $date.ToShortTimeString()
            Write-Output "Mylog: F4 Detected, Exiting the loop at: $mytime"
            $Exit_Command = 'TRUE'
            break
            }
        }
    }
}

The log when running from terminal (and not from startup) (when pressing F3 5 times and F4 one time)
Mylog: Waiting to Keyboard
Mylog: Waiting to Keyboard
Mylog: Waiting to Keyboard
Mylog: Identified Press of key
Mylog: F3 Detected, Pausing Run
Mylog: Waiting to Keyboard
Mylog: Identified Press of key
Mylog: F3 Detected, Resuming Run
Mylog: Waiting to Keyboard
Mylog: Identified Press of key
Mylog: F3 Detected, Pausing Run
Mylog: Waiting to Keyboard
Mylog: Identified Press of key
Mylog: F3 Detected, Resuming Run
Mylog: Waiting to Keyboard
Mylog: Identified Press of key
Mylog: F3 Detected, Pausing Run
Mylog: Waiting to Keyboard
Mylog: Identified Press of key
Mylog: F4 Detected, Exiting the loop at: 7/23/2022 2:35 PM

redpajama_set_name: RedPajamaStackExchange


text:
Rats are everywhere, but rarely studied. One Canadian project that is filling the gap has made disturbing discoveries
Eric McElroy was trundling the garbage bin down his driveway in January of last year when he felt movement on his gloved hand. He watched in horror as four rats erupted from the lid.
After they had fled into the downtown Toronto night, he gathered himself and again started moving the bin. Four more rats burst out.
"At this point, I'm just like aghast," Mr. McElroy recalled. Opening the lid, he saw yet another four. "The rest of them scramble, going north and south, across Bathurst Street, just in every which way. It was really, really terrifying."
While his experience may seem like a nightmare for anyone who takes out the garbage, most urban people have been closer to a rat than they might realize.
Although Alberta claims to be rat-free, these rodents have become firmly ensconced in most cities. The brown rat, which originated in Asia, has followed humans to every continent save Antarctica.
The urban rat is loathed, even feared, but is also one of our most tenacious creations. They are a reflection of our civilization, having learned to live in our shadow and dine off the garbage we generate. And their numbers, according to one of the world's top experts, are jumping globally.
In spite of rats' ubiquity, they aren't studied much, leaving big gaps in our knowledge about them. One of the few Canadian efforts is the Vancouver Rat Project, launched to learn more about the pathogens they carry.
This group has made disturbing findings. The way rats pick up disease-causing micro-organisms prompted the project's head to adopt the descriptor "pathogen sponge." And the research suggests that exterminating some rats caused pathogens to spread more broadly among survivors. But the animal has also elicited a healthy level of respect.
"One of the challenges is that rats are very adaptable, they can live in lots of different situations," said Kaylee Byers, a University of British Columbia doctoral candidate who has worked with the rat project for six years.
"If we want to really evaluate why we have rat infestations, we need to take a look in the mirror at what it is we're doing that's promoting rat infestations."
IMPRESSIVE FECUNDITY
A rash of rat sightings last year in part of downtown Toronto prompted city council to ask staff to look at improving mitigation tactics and education. Their report is due later this year. But any anti-rat measures will be tackling a problem without an easy solution.
Studies show that cats prefer not to tangle with rats. Poisons are not always effective. Few cities have the stomach to loose specially trained dogs on rats and bounties were eliminated decades ago in most jurisdictions. Many exterminators have rebranded as pest-management professionals, in part because the old name carried an unrealistic promise.
One problem is that rats reproduce prolifically.
"Most likely, if you are in New York while you are reading this sentence or even in any other major city in America, then you are in proximity to two or more rats having sex," writes Robert Sullivan in Rats, his book about his observations in a New York alley.
Although estimates vary, experts say a pair of rats will produce around 15,000 descendants in a year. And if a rat population is partly eliminated, survivors will reproduce even more quickly to make up the shortfall.
Bobby Corrigan believes rat numbers have been rising in the cities he visits. The president of RMC Pest Management Consulting in the United States, he is one of the top rat experts in the world and has been working with these rodents for decades.
He suspects the population increases are due to growing urban populations and infrastructure becoming easier to penetrate with age. Warmer winters also may have allowed rats to eke out one extra litter, boosting their already impressive fecundity.
Controlling rats is ultimately about controlling their food supply. Although rats are popularly linked with poverty, they don't discriminate and go where the food is. And they don't need much.
"It only takes a small amount to keep a rat family on your block going; it takes one property owner with a bad garbage can," Mr. Corrigan said. "I think that they're with us forever, because they're so successful at what they do."
'RATS ARE LIKE FEAR'
Not all rats evoke a squeamish response, and some have positive connotations. In his book The Wind in the Willows, Kenneth Grahame portrays the water rat as a loyal and dependable friend to the mole. Domesticated rats have long been kept as pets, including by the author Beatrix Potter.
But wild rats have a different reputation. They tend to make people squirm, perhaps because of the many pathogens they can carry. It was rats that are generally thought to have harboured the bubonic plague that wiped out millions. They are sometimes called "germ elevators" for the way they carry health dangers up and out of the sewers.
"They're also capable, through their ecology of running around, you know, in our backyards and in our alleyways, of picking up these other pathogens that are circulating in human populations," said the Vancouver Rat Project's Ms. Byers.
But rats are not the only animals that can harm us. And yet there's something about this rodent that prompts a visceral distaste.
Dougie Clarke, an expert in poison-resistant "super rats" at Britain's University of Huddersfield, said it might relate to the animal's "snake-like tail" and its habit of entering our personal space.
"A lot of people like squirrels," he said. "They're really tree-climbing rats with fluffy tails … and they carry similar [health risks], but they just tend not to come into our households much."
With construction seasons gearing up across Canada, rats whose burrows are disturbed will soon be looking for new homes. To nest, rats need a void the size of a basketball. They often live underground, but will happily come inside. When they do want to enter a space, they can squeeze through a gap the size of a two-dollar coin.
"Rats are like fear: they creep in at night, in those unseen places of vulnerability," Mr. Sullivan wrote.
The sneaky and dirty image of wild rats has constrained their study. Most people want them removed instead of examined. But while there's much we don't know, those who work with them say they are smart, adaptable creatures capable of learning and empathy.
Rats can swim substantial distances and are clever enough to navigate plumbing and emerge from the toilet. Although this act is sometimes dismissed as a myth, National Geographic uploaded a video in 2015 showing how easily a rat could pull off the trick.
Fifteen years after Paul Lewis found a dead rat filling the bowl of his basement toilet in North Toronto, the image remains vivid in his memory.
"There was no way I was ever going to sit on that toilet seat," he said.
"Every time you opened up the toilet lid it was kind of like, what am I going to see next? Am I going to see a snake? Because I've read those stories too."
https://www.theglobeandmail.com/canada/toronto/article-rats-are-everywhere-but-rarely-studied-one-canadian-project-that-is/
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The Goodman SSZ14 Heat Pump uses the environmentally friendly refrigerant R-410A and features operating sound levels that are among the best in the heating and cooling industry. R-410A is chlorine-free to help prevent damage to the ozone layer. With its 14 SEER rating, the SSZ14 will help reduce energy consumption throughout the life of the system.
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County Court at Law 2: Judge Grace Uzomba defeated Republican incumbent Judge Jason Wolff. Judge Uzomba has spent the better part of her career in private practice.
County Court at Law 7: Judge Michael De Leon defeated Genie Wright. Judge De Leon spent years as a family violence prosecutor. County Court at Law 7 handles family violence cases.
County Court at Law 8: Judge Mary Roman defeated Celeste Brown. Judge Roman spent years as a felony district court judge in the 175th. She retired from that bench in 2016. She has decades of criminal law experience.
County Court at Law 9: Judge Gloria Saldana defeated Wade Shelton. Judge Saldana previously presided over a civil district court bench.
County Court at Law 12: Judge Yolanda Huff defeated Scott Roberts. This court will continue serve as the mental health court.
County Court at Law 13: Judge Rosie Speedlin Gonzalez defeated Crystal Chandler. Like County Court at Law 7, County Court at Law 13 is a specialty court that handles family violence cases.
County Court at Law 14: Judge Carlo Key defeated Susan Skinner. Judge Key previously served as the presiding judge of County Court at Law, but lost in a republican sweep to current Judge Tommy Stolhandske.
County Court at Law 15: Judge Melissa Vara defeated Bob Behrens.
Bexar County Criminal Courts handle misdemeanor cases in Bexar County. This includes arrests made by both Bexar County Sheriff's Office and San Antonio Police Department as well as other local agencies.
If you are arrested in Bexar for a misdemeanor offense, it is important to contact an experienced criminal lawyer immediately. Attorney Dayna Jones has over 11 years handling cases in Bexar County and throughout Texas. Even though misdemeanors are lower level offenses, they still come with harsh penalties and collateral consequences that may negatively affect your life for many years. Contact the Law Office of Dayna Jones for help with your case.


What is the Value of a Child's Life?
I recently recognized that a series of issues I've been struggling with all have one theme in common. The thing that ties each of these issues together boils down to the worth of my children.
Death seems to strip an individual's value from them in the eyes of the world. Daily life moves forward and it's not long before the phrase, "Out of sight, out of mind", applies.
What is the value of one child's life? To their parents, their siblings, their extended family, their circle of friends and acquaintances, to the community they lived in and even to the world at large? It doesn't take a genius to figure out that their value generally diminishes as you go through the list.
The grieving long to have their loved one's worth acknowledged, appreciated and validated beyond the funeral and burial, beyond the first week they return to work, beyond the year of firsts, beyond . . .
This bereaved parent questions her own value as well – to God in particular. I wrote this quite awhile back. It's a prayer of a sort, and deeply personal, but it clearly reflects how circumstances can cause a person to contemplate their significance. Keep in mind that my first child was stillborn, then I lost two in a car accident, and my surviving child has a rare and progressive form of Muscular Dystrophy. I've taken a series of hits.
Value and worth, it's a struggle I see other parents who mourn wrestling with. Support groups, blogs, and Facebook posts are filled with the underlying theme.
Some make a shrine of their child's room. And the outside world shakes their heads in pity – failing to understand why. Honestly, the parent may not be able to put into words why they do it themselves. But their child's possessions are a visual, touchable testimony of both their existence and who they were below the surface. That room and the pictures they treasure, are often all the parents have to hold onto. They've lost their child and cling to the things they loved and touched in their absence.
And really, if you think about the alternative, can you blame them? Does anyone really think about the emotional price a parent pays when they sort through the remnants of their child's life? Do they realize how it feels to decide what to give away – and who to give it to? What to keep. What things most effectively reflect the child they loved. What to throw away; now that, well that's the nauseating one. Disease or accident, murder, suicide, or addiction, military or public service, has snatched their child from their hands and now they feel as if they are choosing to throw away their child, bit by excruciating bit. Maybe the shrine makes more sense now. It's not shameful, it's nothing less than a grieving parent defiantly refusing to toss away the evidence of their child's life. It's all about value and worth.
Almost two years after the collision that killed my daughters, I am still sorting, still deciding what to keep, what to give away – what to throw away. Granted, I was caring for Gracen, but I've had time to complete the task. Every once in awhile, I open the doors to the two rooms that hold the things my children once touched and I make value judgments until my heart can tolerate it no more.
Some parents set up foundations in their child's name for a cause their child was passionate about or to raise funds or awareness for the disease or tragic circumstance that took their child from them. Those foundations meet needs, keep their child's memory alive, and validate their child's worth. And some parents stand jealousy on the sidelines because their child did not live long enough to discover their purpose and passions. There will be no foundation and their child will all too quickly be forgotten, overlooked, or intentionally left out for fear of reminding the grieving parent of their death. Personalized gifts will not include their name, you will be introduced as the parent of one less child – and the parent of a stillborn child will not be asked about their child's birth weight and length; all in the name of compassion. It's not always true that actions speak louder than words. It's amazing how loudly silence speaks.
Polite society encourages the family to let go, move forward, have another baby, take in foster children, adopt, and of course, be thankful for the children you have left; unwittingly conveying the message that the child you lost no longer has significance and that continued grief equates to a lack of appreciation for those you still have.
And the grief-stricken parent fights the war within; attempting to reconcile the worth of their child between the messages they receive from society and the intellectual truth that their child's worth never stemmed from their accomplishments but from the fact that they were theirs and created by God.
The grieving parent is begging – demanding really – that society validates the worth of their child; their contribution, their significance in this world; regardless of their length of life.
I'm not sure any parent passes through the grief process until they either "feel" the validation they crave (because a small group of people do just that) or until they resign themselves to the real truth – that it is enough if they alone recognize the worth of their child in this world. The battle within has been won, the enemy defeated by love – the love of God and the love of the parent. The only thing the parent needs to let go of is the desire to have their child's worth validated by society. However, that's easier said than done. Knowing what needs to be done does not make it easy to do. The heart wants what the heart wants, and it's a process that's mastered one painful step at a time.
This Thanksgiving we will return to Kansas City as we have for the last ten years. Every time we choose to go "home", we pass the accident site twice. In fact, we rarely drive north unless we are heading back to see family and friends. The crosses usher in a heighten level of anxiety for the painful moments that are simply unavoidable. And the return trip heralds in memories of where it all went so wrong. To say I need to emotionally prepare myself for these trips is an understatement of gigantic proportions. But staying home – being alone in our unnaturally quiet house – is exponentially worse.
Gratitude and grief co-exist within my heart. Therefore, this year I plan to allow myself permission to do something I've never permitted myself to do in the past. If I feel overwhelmed, if the sight of healthy intact families, and bright futures pinch just a bit too much, I will slip off and ensconce myself in the room we make use of when we are in town.
I will not make myself be strong when I feel weak as I have done in seasons past. I will not force myself to wear a mask in order to make others comfortable in my presence. I've long been aware that I'm not responsible for how comfortable others feel with my grief, at the same time I've always assumed responsibility for shielding others from my emotions, for their sake and mine. Frankly, an open display of my vulnerabilities is abhorrent to me. In the past I've felt as if leaving the room was just as bad as crying in public. Doing either draws attention and leads people to talk. Now, I'm just tired. I also realize that it's not unreasonable or shameful to remove myself from a situation in which I'm uncomfortable. To remain, to pretend good cheer, that's a burden too great for me to bear and this year – I just don't have the energy.
Deflection, a highly valuable social skill, is not one I've ever become adept at using. Over the years I've been put on the spot and found myself exposing vulnerabilities to family, friends or mere acquaintances to my personal detriment. In the coming weeks I will practice simply not answering a direct question by responding with a question of my own.
Most importantly, I will remind myself of this truth: Honesty does not require transparency; nor does it require vulnerability. It is my right to choose both when and what I feel comfortable sharing and with whom I wish to be transparent and vulnerable.
I don't want attention or pity: I want privacy and understanding. I don't want others evaluating how I'm doing based upon their personal perceptions. If asked, I'll share what I'm comfortable sharing and hope if others later inquire, that no more than what I've shared will be disseminated. Anything more is little more than fodder for gossip.
I will never forget how gutted I felt when I bumped into a friend following the death of my son and she said, 'I heard you weren't doing very well.' What was worse was realizing that the person who reported on my well-being had never asked me how I was doing; they simply watched me and drew their own conclusions. Gossip hurts. While long forgiven, and completely beyond my control, I remain hypersensitive to how I am portrayed to others. It's my reputation and it's my heart that suffers for idle words spoken.
While the things above may seem simple to many, they are challenging for me. So this year, I will keep the commitments I've made to myself and will do my best to let go of the things I can't control. There is grace and forgiveness for those things.
Of the many things for which I'm thankful – the blessings among the thorns, I'm most grateful that I still have David and Gracen. However, I dread that moment that often occurs at Thanksgiving gatherings when asked to share that for which we are thankful. To express my thanks for my husband and daughter draws attention to those who are missing. It's awkward – for me and for everyone else. While grief may overshadow my gratitude, that doesn't mean I don't recognize and appreciate my blessings. So this year, I will give thanks, but I might not have a happy Thanksgiving. Contrary to what the Veggie Tales teach, a grateful heart is not always a happy heart; and that's OK, gratitude is sufficient.


text:
Home » Cat Among the Pigeons
Cat Among the Pigeons
Muirhead, David
Here's another batch of David Muirhead's unrespectable creatures, following his successful earlier volume of hilarious animal accounts (The Bedside Ark ). It offers a wealth of accurate information on each of the profiled creatures, while revealing their softer sides and the near-human frailties from which they suffer – and temptations for which they fall. Delightful, humorous pen-and-ink sketches accompany many of the stories.
Muirhead's mix of humour, mythology, anecdotal tales and folklore builds quirky and captivating portraits of each animal, and makes for a lighthearted, funny – as well as illuminating – read.
This new anthology, offering something different from the standard collection of animal CVs, will appeal to anyone interested in humorous writing and the natural world, no matter their age (from teen to adult) or level of knowledge.
Enjoy these extracts from Cat Among the Pigeons :
Marabou Stork
Long-time English writer and journalist David Muirhead has already published several books, including a collection of satirical short stories that drew acclaim from the British press. Struik Nature published his earlier anthology, The Bedside Ark – A riotous assembly of unrespectable creatures. The Cat Among the Pigeons is his latest release. He lives in Vermont, Western Cape.
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Jansen, Albert
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Every family has its secrets. Fred and Sheila Mercer have worked hard their whole...
Jamie Oliver's Christmas Cookbook
Oliver, Jamie
Jamie's Christmas Cookbook is packed with all the classics you need for the big day and...
But Deliver Us From Evil
Kubuitsile, Lauri
But Deliver Us from Evil tells the story of two young women whose lives converge at a...


He must have slept a long time, for when he awoke he felt wonderfully restored—indeed almost well—and very hungry.
There were voices in the outer cave. Once more, then, it was night; for the goblins slept during the day and went about their affairs during the night.
In the universal and constant darkness of their dwelling they had no reason to prefer the one arrangement to the other; but from aversion to the sun-people they chose to be busy when there was least chance of their being met either by the miners below, when they were burrowing, or by the people of the mountain above, when they were feeding their sheep or catching their goats. And indeed it was only when the sun was away that the outside of the mountain was sufficiently like their own dismal regions to be endurable to their mole eyes, so thoroughly had they become unaccustomed to any light beyond that of their own fires and torches.
Curdie listened, and soon found that they were talking of himself.
'How long will it take?' asked Harelip.
The queen laughed. She seemed in far better humour than the night before.
The king gave a great laugh.
'Better and better!' cried the queen and the prince together, both of them clapping their hands. And the prince made an ugly noise with his hare-lip, just as if he had intended to be one at the feast.
A shoe without a sole.
'What's that horrible noise?' cried the queen, shuddering from pot-metal head to granite shoes.
'I think we had better go to bed,' said the king.
'It's not time to go to bed,' said the queen.
'I would if I was you,' said Curdie.
'Impertinent wretch!' said the queen, with the utmost scorn in her voice.
'An impossible if,' said His Majesty with dignity.
'What a lie!' roared the queen in a rage.
'I will do as I like,' retorted the queen sulkily.
'You ought to do as your own hubby wishes you,' said the king.
'I will not,' said the queen.
'Then I insist upon it,' said the king.
Apparently His Majesty approached the queen for the purpose of following the advice given by Curdie, for the latter heard a scuffle, and then a great roar from the king.
'Will you be quiet, then?' said the queen wickedly.
'I'm going,' said Harelip sleepily.
'So am I,' said the king.
'Oh, no, no, no!' screamed the king in the most supplicating of tones.
Curdie heard only a muttered reply in the distance; and then the cave was quite still.
They had left the fire burning, and the light came through brighter than before. Curdie thought it was time to try again if anything could be done. But he found he could not get even a finger through the chink between the slab and the rock. He gave a great rush with his shoulder against the slab, but it yielded no more than if it had been part of the rock. All he could do was to sit down and think again.
By and by he came to the resolution to pretend to be dying, in the hope they might take him out before his strength was too much exhausted to let him have a chance. Then, for the creatures, if he could but find his axe again, he would have no fear of them; and if it were not for the queen's horrid shoes, he would have no fear at all.
Meantime, until they should come again at night, there was nothing for him to do but forge new rhymes, now his only weapons. He had no intention of using them at present, of course; but it was well to have a stock, for he might live to want them, and the manufacture of them would help to while away the time.


Do you ever have a project that somehow just takes a long time to finish, even though it's in front of you every day?
This is the story of one of those projects around our house...and a message to two dads (mine who lives in Oregon, and Caleb's who is away in the States right now!) to say that it's finally finished!
Five years ago, in May 2006, my dad came out to stay with us for a month. While he was here he noticed something missing at our house.
Through a really cool story (too long to tell...sorry!), while he was here he got word of some money that he had invested 40 years before, that was finally paying off. With much love for us, he offered to use part of it to buy something that would meet that need.
A garden storage house for our yard! After looking at various stores in the area, we found just the right one to fit our needs.
And put it together on the spot, like a giant Lincoln Log project!
It looked so cute when it was done - like having a big playhouse in the yard.
Later that summer, we spent a Saturday staining it as a family.
What a project that was, getting two full coats on - though we did have some pretty awesome helpers to get it done!
Years went by, and still no roof tiles - it was one of those things we kept meaning to do, but somehow it was never in the budget to actually purchase them. The garden house worked (it holds so much stuff that would otherwise be crowded into the garage), and didn't even leak too much as it had a tar paper roof. But we knew that some day we'd have to put some tiles on it.
I'm not even sure when the next part of the story happened...maybe two years ago? A friend of ours gave us some roof tiles from his parents house which Dave and Caleb put on. Unfortunately, there weren't enough, so only half the roof was done.
It sat for another year with a partial roof of tiles.
But finally this week...Caleb went and picked up the last tiles...more than five years after this project was started!
He headed up to the roof and I waited on the ladder for the last nail to go in!
Ta-dah!!! The roof is finally finished dad!
Glad to have that project finally checked off our "To Do" list!
Thanks SO much dad for making that investment in the garden house for us!! I think of you every day when I see it!
Wow! How'd you find those old pictures?? What a fun blog post!


I guess this is good bye, since all of my stories are gone. I have no reason to come back. So everybody that read my stories thank you!
No way to repost :/ ?
A lot of us lost a bunch of threads after the recent fudge up on the site, it sucked but isn't the end of the world.
Can't you repost the stories you lost?
No I didn't save them. One story had over 91,000 hits I've worked over 2 years on it! I'm thought about it for over 2 days. I came back, still sick about what happened but not ready to leave yet.