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Updates on our life at Hill Farm Bed and Breakfast in the Derbyshire Dales - why not come and stay with us to experience it for yourself! Find out more at www.hillfarmderbyshire.co.uk
Thursday, 31 March 2016
Wednesday, 30 March 2016
Worried sheep ..
Social ME-dia
Twitter and Facebook as well as being listed with Britain's Finest as you'll see from the logo on our website. Check our entry on their site to discover our exciting opening offer ....
Sleet, snow and a rainbow
Tuesday, 29 March 2016
Britain's next top model
Sunday, 27 March 2016
Traditions at Easter
Saturday, 26 March 2016
Tulips from ....
Baking bunnies
Thursday, 24 March 2016
Naughty girls - again
Easter welcome
Wednesday, 23 March 2016
Man-ual labour?
Monday, 21 March 2016
Ironing and duvet flipping
'Our' flock arrive
Sunday, 20 March 2016
Hot air balloon
Saturday, 19 March 2016
Man-ual labour
How posh
Friday, 18 March 2016
After a day with my sister I have finally bitten the bullet and set up a Twitter account for our B&B, you can find me @HillFarmBnB and I promise (possibly) that my future tweets won't all be sheep related!
A 'new' gate
Thursday, 17 March 2016
More sheep
ewenique' flock at the Country Living Spring Fair. Deciding to buy one of these amazing hand crafted footstools is the easy decision, the challenging part is working out which one to take home as all are individuals with faces carved from English Lime, fleeces that come from rare breed sheep and then there is the name .... . I also went to an excellent talk by Fiona Sloan about 'how to set up and manage a small flock of sheep' which sounds an attractive proposition despite everyone telling me that sheep are four legs looking for ways to kill themselves! If you feel a 'ewenique' sheep is an easier option the Fair is on until Sunday.
Monday, 14 March 2016
Red sky at night ..
Someone stopped at the farm to say the sheep were out on the lane, when I went to investigate they turned out to be the newly arrived black headed ones and although they were standing obligingly next to their gate, once I opened it they decided to head off and explore. Their explorations left an easy to follow trail (!) and I found them in a neighbours field with her horses. Fortunately at that moment George drove by in his land rover so I was able to flag him down, he keeps the very well behaved sheep in the field opposite us. With his help we drove the sheep back home, but as we couldn't see any obvious escape route and as by now it was getting dark it is probably only a matter of time before they get out again.
Hedge maintenance
Bricklayer Dave
Sunday, 13 March 2016
Washing and drying
Saturday, 12 March 2016
Bird song
Working outside these last few days I've been surrounded by the sounds of birds calling. Earlier I heard a microlight overhead and when I looked up I spotted three buzzards wheeling high and silently way above it on the thermals.
Friday, 11 March 2016
Misty sun
Another project
Thursday, 10 March 2016
Cat on a hot tin roof
An audience
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The company that developed an aerial ignition system that can be carried by a drone has introduced an improved model that can hold almost three times the number of plastic spheres.
The Ignis 2.0 made by Drone Amplified can be loaded with 400 to 450 spheres that ignite 30 to 45 seconds after being released from the drone. Their previous system, Ignis 1.0, carried 150 spheres. The new design is easier to maintain and can drop the spheres at up to four times the rate if desired, an increase from 30 to 120 spheres per minute. By using an Android app, the user can configure ignition spacing, number of ignition spheres, mission duration, and altitude.
Firefighters have employed the concept of using machines for aerial ignition for 40 to 50 years starting with an aerial drip torch suspended below a helicopter and later advancing to equipment installed in the open door of a helicopter.
Just before they are released, the spheres, which contain a chemical, are injected with a second chemical that causes them to ignite 30 to 45 seconds later. Aerial ignition allows prescribed fires or firing operations on wildfires to be ignited in areas that can be difficult for firefighters on the ground to reach safely, reducing their exposure to hazards. It can also ignite controlled burns more quickly than it can be done by personnel on foot, and at less cost than a helicopter.
The Department of the Interior began experimenting with drones for aerial ignition in 2017 and in 2018 began using a much larger aircraft, the Matrisse 600 that can carry up to 13 pounds. In August it was used to ignite a firing operation at night on the Inyo National Forest on the Springs Fire 13 miles southeast of Lee Vining, California.
According to Drone Amplified, the DOI just finished testing the new Ignis 2.0 in Arizona and ordered 20 for immediate delivery.
The Chief Engineer for Drone Amplified, Jim Higgins, was a mechanical engineering graduate student at the University of Nebraska Lincoln when he and others built the first drone to be used to ignite a prescribed fire at Homestead National Monument west of Beatrice, Nebraska. Drone Amplified is based in Lincoln, Nebraska.

subset: commoncrawl


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Willamette Valley, Oregon
Available Terragena wines from this vineyard: 2016 Oregon Pinot Gris
Owned and cared for by Pete and Marcia Buffington, this vineyard is located in the Willamette Valley near Scotts Mills, Oregon, and near the beautiful Abiqua Falls. The Abiqua Wind vines were planted on own-roots in 1978, so they are very mature at this point. We are partnering with this excellent vineyard to make a 2016 Willamette Valley pinot gris. Unfortunately, due to the odd laws around labeling wine, because we made this wine in California, we are unable to put the name of the vineyard or the Willamette Valley region on the wine bottle label. However, all grapes for this wine were sourced from the Abiqua Wine Vineyard.


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author: Ian Fleming
name: Matthew
average rating: 3.77
book published: 1958
rating: 4
read at: 2018/08/27
date added: 2018/08/27
Fleming's Bond may have gotten in over his head in this Jamaican outing. After sufficient rest following a near-fatal wound, Bond is sent "on a holiday in the sun" to investigate the mysterious disappearance of his friend Commander Strangways (from Live and Let Die). This book serves as sort of a "reset" for the Bond character, miraculously surviving a fatal blow at the end of From Russia With Love. Bond is rusty, careless, and seemingly fed-up with his lot, but he grudgingly flies off to one of the last vestiges of the crumbling British empire, reconnects with his friend Quarrel and ruminates about the time that has passed since his last visit to the island.
Dr. No is a love letter to Fleming's Jamaica, replete with exquisite detail of the high and low societies of the tropical island where he spend so much time writing. Bond, like Fleming, grudgingly picks up his next job and re-familiarizes himself with the tropes that made him great. This is the return of the great adventure novel in all its swashbuckling glory–a mystery, a megalomaniacal villain, and solitary dangers to test our hero to his very core. The climactic battle, though, is not with the titular baddie, but with something even more typical of the genre (and done with a self-aware flourish) that serves as a new direction for the Bond series. It's not the best in the bunch, but it's certainly worth a read!
Pick up a copy on Amazon (affiliate link)


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In this special edition of App Industry News, we look at all the news Google has been making this week. That includes some perspective on Google's new Apple-bashing star, what Google TV means for the Android OS, and what you can expect from the coming Android update. Why the world needs Android, according to Google […]
In this special edition of App Industry News, we look at all the news Google has been making this week. That includes some perspective on Google's new Apple-bashing star, what Google TV means for the Android OS, and what you can expect from the coming Android update.
Why the world needs Android, according to Google
A star is on the rise at Google, an executive unafraid to publicly rip the search giant's hottest competitor — Apple. His name is Vic Gundotra and he's Google's vice president of engineering. He gave the Thursday keynote at Google's I/O event, and spent much of his speech ripping Apple. (Do you think Eric Schmidt was smiling? Or perhaps pulling the puppet strings?)
That last line is probably not fair but Gundotra was not shy while he talked (ripped?) about Apple. Gundotra is a smooth, captivating public speaker and clearly a fine voice for Google. And yes, a bit melodramatic. He kicked off his speech (after the usual pleasantries) talking about how consumers faced "a Draconian future" controlled by one choice and one device (as a background video flashed an unflattering picture of Steve Jobs) if Google didn't embrace and push Android. "That's a future we don't want," he said, as the crowd applauded so enthusiastically one would think Jobs was talking. "If you believe in innovation from everyone, than welcome to Android."
In VentureBeat, Paul Boutin notes that Gundotra spent much of his carrier at Microsoft, "where he got software developers to put down their seething hatred for anything Bill Gates touched and actually try Windows Live." There is even speculation that Gundrota could be the next top executive at Google, a notion Boutin doesn't see as likely. "But he's certainly the guy to put in front of the geeks," he writes.
Gundotra is smooth. Take a look.
Here comes Google TV. Will we watch?
The Android OS is of course on many, many phones and it will arrive very soon on new netbooks. Now, Android will be coming to your living room TV as well thanks to what's called Google TV.
It is a logical step, of course, as makers of slick new HDTVs and Blu-ray players have been adding wireless Internet connections that allow people to stream content onto thier TVs. Heck, I have two devices in my living room that already allows me to watch streamed Netflix movies — a $150 Insignia (Best Buy's house brand) Blu-ray player and a Wii.
But Google TV promises to be a more robust offering than what we've seen so far in the connected TV space. For one, it puts the Chrome browser and Android OS on your TV, accessed through an HDMI cable and special Google set-top box. You'll be able to stream content from sites like Hulu, YouTube (duh!) and Bob's family website, where you can watch as Bob uploads cute videos of his kids.
Apparently, the demo at the I/O conference wasn't very smooth and it led to more questions about the service than answers. Sony, Intel and Best Buy all signed on as partners, but a release date for the product remains unclear at the moment.
Android update coming soon
Finally, Google said that version 2.2 of the mobile Android OS will be available soon. Dubbed Froyo (huh, why not Frodo?), the update will allow for device tethering and full Flash support. (Insert Steve Jobs jab here.)
What's unclear is whether or not existing devices will be able to upgrade to the new software. The growing cadre of HTC fan boys (and, um, I'm raising my hand) have started to speculate on which HTC phones will get the upgrade. The feeling is that all 2010 models — such as the Incredible (Verizon) and Evo (Sprint) — will get the upgrade, but no one knows for sure when that will happen.


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Miami hip-hop band ¡Mayday! drop this new track today, "Lifetimes," produced by Danny "Keys" Perez of The Pushers and Frank A. Vasallo. "Lifetimes" is the third release off ¡Mayday!'s upcoming album, South Of 5th, due out September 7. . ¡Mayday! will also be heading out on a national tour beginning September 7. @maydaymusicRead more
After the singles "Static" and "All Day Long", the Inland Empire/Los Angeles Hip-Hop artist Trizz, drops the final single before his new album "The Attic" comes out on July 13th. The new single "86" is featuring T.F and it's produced by Stone's Neighbor. Trizz says "My homie T.F told me sometimes you just got to […]Read more
Rebel Diaz – Y Va Caer ( Feat. Ana Tijoux ) First single off of upcoming album América -vs- Amerikkka by Rebel Diaz, due Summer 2018. Primer adelanto del nuevo disco América -vs- Amerikkka, de Rebel Diaz, que se estrena este Verano 2018. @RebelDiaz @AnaTijouxRead more


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- Research
- Open Access
- Published:
Drug metabolite synthesis by immobilized human FMO3 and whole cell catalysts
Microbial Cell Factories volume 18, Article number: 133 (2019)
943 Accesses
1 Citations
- The Publisher Correction to this article has been published in Microbial Cell Factories 2020 19:78
Sufficient reference standards of drug metabolites are required in the drug discovery and development process. However, such drug standards are often expensive or not commercially available. Chemical synthesis of drug metabolite is often difficulty due to the highly regio- and stereo-chemically demanding. The present work aims to construct stable and efficient biocatalysts for the generation of drug metabolites in vitro.
In this work, using benzydamine as a model drug, two easy-to-perform approaches (whole cell catalysis and enzyme immobilization) were investigated for the synthesis of FMO3-generated drug metabolites. The whole cell catalysis was carried out by using cell suspensions of E. coli JM109 harboring FMO3 and E. coli BL21 harboring GDH (glucose dehydrogenase), giving 1.2 g/L benzydamine N-oxide within 9 h under the optimized conditions. While for another approach, two HisTrap HP columns respectively carrying His6-GDH and His6-FMO3 were connected in series used for the biocatalysis. In this case, 0.47 g/L benzydamine N-oxide was generated within 2.5 h under the optimized conditions. In addition, FMO3 immobilization at the C-terminal (membrane anchor region) significantly improved its enzymatic thermostability by more than 10 times. Moreover, the high efficiency of these two biocatalytic approaches was also confirmed by the N-oxidation of tamoxifen.
The results presented in this work provides new possibilities for the drug-metabolizing enzymes-mediated biocatalysis.
Human FMO3 is an important non-cytochrome P450 drug metabolizing enzyme in adult human liver, catalyzing the monooxygenation of a wide variety of nucleophilic heteroatom-containing drugs, xenobiotics and dietary compounds to their corresponding N-oxide metabolites [1]. In the course of drug discovery and development process, sufficient reference standards of drug metabolites are required for their pharmacologic and toxicologic characterization [2]. However, such drug standards are often expensive or not commercially available, particularly in the case of preclinical/clinical or new therapeutic drugs. Chemical synthesis of drug metabolites is often difficulty due to the highly regio- and stereo-chemically demanding [3]. In addition, although drug metabolites can be generated directly by drug metabolizing enzymes in vitro with high selectivity, the synthesis on preparative scale is challenging due to low catalytic activity/protein stability, difficult re-use of the enzyme, and requirement for the expensive cofactor(s). These drawbacks can often be overcome by whole cell catalysis due to advantages like low cost, large scale application and cofactor regeneration [4,5,6], as well as enzyme immobilization due to advantages such as improved enzyme thermostability, feasible enzyme recovery/recycling, and straightforward downstream processing [7, 8].
Benzydamine is a nonsteroidal anti-inflammatory drug [9], and primarily converted into N-oxide metabolite by FMO3 in human liver. The N-oxygenation of benzydamine as a marker reaction can reflect FMO3 activity [10, 11]. In this work, using benzydamine as a model drug, two approaches were tested and optimized for the synthesis of benzydamine N-oxide: an enzyme immobilization system using two HisTrap HP columns connected in series carrying His6-GDH and His6-FMO3 enzymes respectively, and a two-strain-mixed-culture strategy using cell suspensions of E. coli JM109 (containing a FMO3 gene) and BL21 (containing a GDH gene). GDH-mediated glucose oxidation is used for recycling the cofactor NADPH (Scheme 1).
Results and discussion
Purification and spectroscopic characterization of FMO3 and GDH
FMO3 and GDH were successfully expressed in E. coli and purified by Ni-affinity chromatography. Subsequently, spectroscopic characteristics of the purified proteins were determined. UV–vis absorption spectra show three maxima around 280, 375 and 450 nm for the purified FMO3, and one maximum around 280 nm for the purified GDH (Additional file 1: Fig. S1a, b). The concentration of holo-FMO3 (168.3 μM, determined by absorbance at 450 nm) accounts for 56% of the total FMO3 (both holo- and apoprotein, 300.3 μM, determined by Bradford assay). In addition, the SDS-PAGE (Additional file 1: Fig. S1c) shows a band around 58 kDa for FMO3 and 30 kDa for GDH in good agreement with their molecular weights calculated from the amino acid sequences.
Catalytic properties of FMO3 and GDH
Subsequently, the catalytic properties of the purified enzymes were characterized in term of ion strength, pH, pH stability, temperature and thermostability. Figure 1a, b shows that the highest activity of FMO3 is achieved at pH 8.1 with an optimized ionic strength of 0.535 M, and this enzyme is stable in alkaline environments (Fig. 1c). In addition, the optimum temperature of FMO3 activity is 40 °C (Fig. 1d), and a relatively weak activity is observed at temperatures below 25 °C. The half-life of FMO3 at 40 °C is determined based on the formula of T1/2 = ln2/kd, where kd is calculated from the slope of the plot ln (residual activity) versus time [12]. Figure 1e, f and Table 1 show that FMO3 has a half-life of 78 min at a low preincubation concentration (2 μM). However, this enzyme is unstable and totally inactivated within 20 min at a high preincubation concentration (25 μM). For the characterization of GDH-mediated glucose oxidation, Fig. 2 shows an optimal pH of 8.10 and temperature of 15 °C. Unlike FMO3, GDH is stable in acidic environments and has a relatively high activity at low temperatures.
Immobilization of enzymes
Following enzymatic characterizations of the purified FMO3 and GDH, enzyme immobilizations were carried out. The His6-tagged FMO3 (with a total activity of 693 mU) and GDH were separately loaded onto two different HisTrap HP columns (1 mL) at a flow rate of 0.5 mL/min by using a peristaltic pump. Immobilization was completed as soon as the enzyme solution was passed through the affinity column (enzyme purification and immobilization can be combined here). The immobilization yield was determined by measuring the total residual enzyme activity in the flow-through [13]. Table 2 shows that more than 99% of immobilization yield is achieved for both GDH and FMO3.
Optimization of the immobilized-enzyme system
Subsequently, the two enzyme columns were connected in series used for the biotransformation (Additional file 1: Fig. S2). The experiments were performed in a Stuart incubator, and the effects of pH, temperature, histidine and glycerol addition on the substrate conversion were investigated (Figs. 3 and 4). Figure 3a, b shows that the highest activity was obtained at pH 8.4 and 40 °C. In addition, FMO3 immobilization significantly increased its thermostability by more than 10 times (Fig. 3c, d and Table 1). If the inactivation of FMO3 was induced by intermolecular hydrophobic interaction between the C-terminal (membrane anchor region) [14], the improved thermostability of FMO3 after affinity immobilization, would be readily rationalized.
Another parameter to be optimized was the histidine concentration in substrate mixture. Although a certain concentration of histidine in solution can decrease non-specific interactions of enzymes to the affinity column [15, 16], the activity of immobilized FMO3 was not improved after the addition of histidine (Fig. 4a). Similar results were also observed for the addition of glycerol in the substrate mixture (Fig. 4b).
Synthesis of benzydamine N-oxide by the immobilized-enzyme system
Following the optimization of the different parameters, the GDH-FMO3 columns were used for the biotransformation of benzydamine to its N-oxide metabolite. Table 2 shows an activity recovery of 46% and 37% for the enzyme immobilization of GDH and FMO3 respectively. Figure 5 shows that more than 97% conversion (above 0.47 g/L N-oxide product) was reached within 2.5 h under the optimized conditions.
Synthesis of benzydamine N-oxide by the optimized whole cell system
Another approach is whole cell catalysis. A two-strain-mixed-culture system was tested for the biotransformation of benzydamine to its N-oxide metabolite. Effects of the ratio of two cell suspensions (harboring either FMO3 or GDH), total cell density, citrate, substrate and NADP+ concentration on biotransformation was investigated (Fig. 6). Figure 6a, b show that the highest conversion is achieved at a FMO3/GDH ratio of 3 with a total cell concentration of 40 gwcw/L. Although citrate was previously proved to accelerate the NADPH regeneration within E. coli [17], its addition did not improve the biotransformation here (Fig. 6c). Moreover, substrate inhibition was observed at a high concentration (above 8 mM) of benzydamine (Fig. 6d), and the addition of 5 mM NADP+ gave the highest FMO3 activity (Fig. 6e). Under the optimized conditions, more than 98% conversion (above 1.2 g/L N-oxide product) was reached within 9 h (Fig. 7). Whole cell catalysis was also performed in the absence of GDH but presence of FMO3, and only limited benzydamine N-oxide (< 5% conversion yield) was generated within 24 h. Cells harboring the empty vector (in the absence of FMO3) served as negative control and as expected negligible benzydamine conversion (< 1%) was observed within 24 h.
In the specific case of FMO enzymes, Hanlon and co-workers have successfully applied the FMO3-based whole cell catalysis for the conversion of Moclobemide to its N-oxide metabolite with a product titer of 0.086 g/L within 24 h [17]. In addition, in another case of FMO2-based whole cell catalysis, 100% conversion (around 0.325 g/L product titer) of 1 mM benzydamine was reached within 16 h [18]. In both cases above, the addition of NADP+ and citrate was used for the NADPH regeneration. For the enzyme immobilization, Ramana and co-workers have successfully improved the stability of FMO3 by immobilizing this enzyme on magnetic nanoparticles using glutaraldehyde as cross-linker. The immobilization of this enzyme onto the glutaraldehyde-coated nanoparticles was instantaneous, with an immobilization yield of 100% [19]. FMO3 has also been successfully immobilized on electrode surfaces, in order to be used for electrocatalysis or developing electrochemical biosensors [20].
The approaches described herein provide new possibilities for the FMO3-mediated biotransformation. Affinity immobilization of the membrane anchor region of FMO3 leads to significantly increased thermostability, providing a strategy for biotechnological applications of membrane-bound proteins and drug-metabolizing enzymes.
Synthesis of tamoxifen N-oxide by the two easy-to-perform approaches
In order to further confirm the efficiency of the two biocatalytic systems. Another FMO3 substrate, tamoxifen, was tested. The results showed that more than 95% of substrate conversion (0.19 g/L N-oxide product) was obtained within 2 h using the immobilized enzyme system (Fig. 8a). The whole cell catalysis showed 77% (0.30 g/L N-oxide product) and 100% (0.38 g/L N-oxide product) of substrate conversion within 8 and 24 h respectively (Fig. 8b).
Although high conversion yields were obtained for the N-oxidation of both benzydamine and tamoxifen in this work, substrate solubility and substrate/product inhibition should be tackled in the future. For FMO3, majority of its known substrates are hydrophobic compounds with low solubility or even insolubility in aqueous media, which greatly limits the enzymatic efficiency. One solution to this limitation is the use of water miscible or immiscible organic solvents to develop mono- or biphasic media for efficient biocatalysis. Water miscible solvents are generally used to facilitate the substrate/product solubility and to improve the reaction rates whereas water immiscible solvents can regulate the distribution of toxic substrates/products around the enzyme, relieving substrate or product inhibition [21, 22]. In addition, overexpression of FMO3 in E. coli is another challenge which need to be tackled in order to further improve the catalytic efficiency of whole cell catalysts. Human FMO3 is a membrane associated protein. Overexpression of this protein is limited by the membrane's capacity. C-terminal truncation of FMO3 can be a possible strategy to remove membrane-bound anchor and produce soluble cytosolic proteins.
In summary, two easy-to-perform approaches were successfully applied for synthesizing FMO3-generated drug metabolites, and both methods lead to high substrate conversions. Future work will focus on relieving substrate inhibition during the whole cell catalysis.
Materials and chemicals
2-Mercaptoethanol, Acetonitrile, Ampicillin sodium salt, Benzydamine hydrochloride, Benzydamine N-oxide hydrogen maleate, Bradford Reagent, d(+)-Glucose, Flavin adenine dinucleotide disodium salt hydrate (FAD), HisTrap HP columns, IGEPAL CA-630, Imidazole, Kanamycin, Nickel(II) sulfate hexahydrate, Lysozyme from hen egg white, Phenylmethylsulfonyl fluoride, and potassium phosphate were purchased from Sigma-Aldrich. NADPH tetra(cyclohexammoninum) and β-NADP sodium salt were purchased from Carbosynth. Isopropyl-beta-d-thiogalactopyranoside (IPTG) was purchased from BIOSYNTH. TRIS Base was purchased from Fisher Molecular Biology. Yeast Extract and Tryptone were purchased from Fisher Bioreagents. PageRuler Unstained Protein Ladder was purchased from Thermo Fisher Scientific Baltics UAB.
Protein expression
Human FMO3 gene with C-terminal His6-tags inserted into pJL2 was expressed in E. coli JM109 [23], and glucose dehydrogenase (GDH) constructed in pET28a was expressed in E. coli BL21 [24]. After 24 h post-induction, the cells were harvested by centrifugation at 4500×g, washed twice with 0.1 M phosphate buffer (pH 7.0), and stored at − 20 °C.
Protein purification
For the purification of FMO3, cell pellets were resuspended in lysis buffer (20% glycerol, 5 mM β-mercaptoethanol, 0.5 mM PMSF, 0.5 mg/mL lysozyme in 50 mM potassium phosphate buffer, pH 7.4), and stirred at 4 °C for 1 h, followed by ultrasonication. The lysate was ultra-centrifuged at 41,000 rpm for 1 h at 4 °C. The resulting cell debris was resuspended in 1% IGEPAL CA-630, and stirred for 2 h at 4 °C, followed by ultra-centrifuged at 41,000 rpm for 1 h at 4 °C. The resulting supernatant was loaded onto a DEAE ion exchange column followed by Ni-affinity chromatography, and the target proteins were eluted by application of 40 mM Histidine. Eluted fractions detected by spectrophotometer were collected and buffer exchanged to storage buffer (100 mM phosphate buffer at pH 7.4, 20% glycerol and 1 mM EDTA) by 30 kDa cutoff Amicon membranes. The purified protein was analyzed by 12.5% SDS-PAGE and stored at − 80 °C. The holo-protein concentration was determined spectrophotometrically using the absorbance at 450 nm (with a molar extinction coefficient of 11,300 M−1 cm−1) [25]. The concentration of total protein (both holo- and apo-protein) was determined by Bradford assay.
For the purification of GDH, cell pellets resuspended in 0.1 M phosphate buffer (pH 7.4) were disrupted by sonication, followed by centrifugation at 12,000×g for 20 min at 4 °C. The resulting supernatant was loaded onto a nickel affinity column. Subsequently, the column was washed by washing buffer 1 (50 mM imidazole, 50 mM sodium phosphate buffer, pH 7.0, 300 mM NaCl), and washing buffer 2 (the concentration of imidazole was increased to 100 mM). The 6× His tagged proteins were eluted with 250 mM imidazole. The eluted solution containing target proteins was buffer exchanged to the storage buffer (0.1 M phosphate buffer, pH 7.0, 50% glycerol) by 30 kDa cutoff Amicon membranes, analyzed by 12.5% SDS-PAGE and stored at − 80 °C. The protein concentration was determined by Bradford assay.
The effect of ionic strength on FMO3 activity
The optimal condition for FMO3-mediated catalysis was investigated, as well as GDH-mediated glucose oxidation.
First of all, the effects of ionic strength on FMO3-mediated catalysis was investigated. The reaction mixture contained 0.3 μM FMO3, 0.5 mM benzydamine (the substrate), 0.5 mM NADPH, in 0.01–0.5 M of potassium phosphate buffer (pH 7.4) with a total volume of 200 μL. The corresponding ion strength of the reaction solution was determined according to https://www.liverpool.ac.uk/pfg/Research/Tools/BuffferCalc/Buffer.html as shown in Table 3. The reaction was initiated by addition of NADPH and incubated at 35 °C for 15 min before termination by the addition of 200 μL ice-cold acetonitrile. The resulting mixture was centrifuged at 12,000×g for 5 min, and 100 μL of the supernatant was analyzed by HPLC equipped with 4.6 × 150 mm 5 μm Eclipse XDB-C18 column at room temperature with the UV–visible detector set at 308 nm. For the separation of benzydamine and its N-oxide, a mobile phase of 22% acetonitrile and 78% formic acid (0.1%) in water was used at a flow rate of 0.5 mL/min.
pH dependence of FMO3
The optimal pH of FMO3-mediated benzydamine N-oxidation was investigated at different pH with a constant ion strength of 535 mM. The buffers were prepared according to https://www.liverpool.ac.uk/pfg/Research/Tools/BuffferCalc/Buffer.html. The reaction mixture contained 0.6 μM FMO3, 0.5 mM benzydamine, 1 mM NADPH, in buffers of different pH. The reaction mixture was incubated at 35 °C for 15 min before terminated by 200 μL ice-cold acetonitrile. The sample was analyzed by HPLC as described above.
pH stability of FMO3
The pH stability of FMO3 was performed by incubating the enzyme (with a concentration of 6 μM) in different pH buffer for 12 h at 4 °C. The residual activity of FMO3 was determined at 35 °C, with a reaction mixture of 0.6 μM FMO3, 1 mM NADPH, 0.5 mM benzydamine, 0.2 M potassium phosphate buffer (pH 8.1), in a final volume of 200 μM. The reaction was stopped by 200 μL ice-cold acetonitrile and analyzed by HPLC as described above.
Optimal temperature and thermostability of FMO3
The optimal temperature of FMO3-mediated catalysis was determined by measuring the activity at different temperatures ranging from 5 to 50 °C. The thermostability of FMO3 was determined by incubating the enzyme (with a concentration of 2 μM and 25 μM) at 40 °C for 3 h. Samples were taken at different time-points, followed by measuring residual activity at 35 °C as described above.
Optimal pH and pH stability of GDH
Optimal pH of GDH-mediated catalysis was determined by using different pH buffer as described above (with a constant ion strength of 535 mM). The reaction mixture consisted of 0.2 μM GDH, 0.2 mM NADP+, 1 mM glucose, in different pH buffer with a total volume of 200 μL. The generated NADPH was monitored spectrophotometrically using the absorbance at 340 nm (with a molar extinction coefficient of 6, 220 M−1 cm−1). The pH stability of GDH was determined by pre-incubating the enzyme (with a final concentration of 2 μM) in different pH buffer for 24 h at 4 °C, followed by measuring residual activity at 25 °C in buffer of pH 8.1.
Optimal temperature of GDH
The optimal temperature of GDH-mediated catalysis was determined by measuring the activity at different temperatures ranging from 5 to 40 °C. The reaction mixture consisted of 0.2 μM GDH, 0.2 mM NADP+, 1 mM glucose, in 0.2 M phosphate buffer (pH 8.1) with a total volume of 200 μL. The activity assay of GDH was performed by using spectrophotometer as described above.
Enzyme immobilization—preparation of enzyme binding columns
The HisTrap™ HP column (1 mL, precharged with Ni2+ ions) washed with 100 mL of 0.2 M phosphate buffer (pH 8.1) was used for the enzyme immobilization. The His6 tagged GDH and FMO3 were separately immobilized onto two different HisTrap™ HP columns.
For the first column, 5 mL of GDH solution (0.48 mg/mL, 16.8 μM) was loaded onto a HisTrap™ HP column (1 mL) at a flow rate of 0.5 mL/min by using a peristaltic pump. Subsequently, the column was washed with 30 mL of 0.2 M phosphate buffer, pH 8.1. For the enzyme immobilization onto the second column, 5 mL of FMO3 solution (0.3 mg/mL, 5 μM) was used with the same immobilization procedure. Subsequently, the two columns were connected in series used for the following analyses.
Enzyme immobilization—optimal pH
The connected enzyme columns were equilibrated with 8 mL of a substrate solution (1 mM NADP+, 1.5 mM glucose, 0.5 mM benzydamine in buffers of different pH) at a constant flow rate of 1 mL/min. 200 μL of flow-through mixed with equal volume of acetonitrile was analyzed by HPLC. The reaction was carried out at 25 °C in an incubator (Stuart orbital incubator SI50).
Enzyme immobilization—optimal temperature
The connected enzyme columns were equilibrated with 8 mL of a substrate solution (1 mM NADP+, 1.5 mM glucose, 0.5 mM benzydamine in 0.2 M phosphate buffer, pH 8.1) at a constant flow rate of 1 mL/min before analyzing the flow-through by HPLC. The reaction was carried out at different temperatures ranging from 20 to 45 °C in a Stuart incubator.
Enzyme immobilization—thermostability
The reaction was carried out at 40 °C in a Stuart incubator. The connected enzyme columns were equilibrated with 8 mL of a substrate solution (1 mM NADP+, 1.5 mM glucose, 0.5 mM benzydamine in 0.2 M phosphate buffer, pH 8.1) at a constant flow rate of 0.5 mL/min. Subsequently, 200 μL of flow-through solution was collected at different time point (0, 20, 40, 60, 80, 120 min), and analyzed by HPLC as described above.
Enzyme immobilization—the effects of glycerol and histidine in substrate mixture on biotransformation
The connected enzyme columns were equilibrated with 8 mL of a substrate solution (1 mM NADP+, 1.5 mM glucose, 0.5 mM benzydamine, 0–12% glycerol or 0–6 mM histidine, in 0.2 M phosphate buffer, pH 8.1) at 25 °C. Subsequently, 200 μL of flow-through solution was collected and analyzed by HPLC.
Immobilized GDH-FMO3 mediated biocatalysis generating benzydamine N-oxide under optimized conditions
15 mL of substrate solution (1 mM NADP+, 4.5 mM glucose, 1.5 mM benzydamine, in 50 mM Tris–HCl, pH 8.4) stirred by a magnetic bar was circularly flowed through (1 mL/min) the two enzyme columns by using a peristaltic pump at 35 °C. 100 μL of substrate solution was taken every 30 min, mixed with 100 μL acetonitrile, and centrifuged at 12,000×g for 5 min. The resulting supernatant was analyzed by HPLC.
Whole cell catalysis—ratio optimization of GDH-BL21 and FMO3-JM109
The E. coli JM 109 harboring FMO3 and E. coli BL21 harboring GDH were used for the whole cell catalysis. The ratio of FMO3-JM109 and GDH-BL21 in reaction mixtures was optimized. The reaction mixture contained different amounts of two cell suspensions (Table 4), 1 mM NADP+, 10 mM glucose, 1 mM benzydamine in 0.2 M potassium phosphate (pH 8.1) buffer with a total volume of 1 mL (in a 50 mL falcon). The reaction was carried out at 35 °C, 200 rpm for 40 min, and terminated by addition of 1 mL ice-cold acetonitrile. The resulting solution was centrifuged at 12,000×g for 5 min and analyzed by HPLC as described above.
Whole cell catalysis—optimization of cell concentration
The optimization of cell concentration was performed by incubating different amounts of cell suspension (Table 5), 1 mM NADP+, 10 mM glucose, 1 mM benzydamine, in 0.2 M potassium phosphate buffer with a total volume of 1 mL. The reaction was carried out at 35 °C, 200 rpm for 40 min, terminated by 1 mL of ice-cold acetonitrile and analyzed by HPLC as described above.
Whole cell catalysis—the effects of citrate addition
The effects of citrate on FMO3-based whole cell catalysis was explored. The incubation mixture consisted of 30 gwcw/L FMO3-JM109, 10 gwcw/L GDH-BL21, 1 mM NADP+, 10 mM glucose, 1 mM benzydamine, different concentration of citrate (0–100 mM), in phosphate buffer (pH 8.1) with a total volume of 1 mL. The reaction was carried out at 35 °C, 200 rpm for 40 min, and analyzed by HPLC as described above.
Whole cell catalysis—optimization of substrate concentration
The reaction mixture contained 30 gwcw/L FMO3-JM109, 10 gwcw/L GDH-BL21, 1 mM NADP+, 10 mM glucose, different concentration of benzydamine ranging from 1 to 10 mM, in phosphate buffer (pH 8.1) with a total volume of 1 mL. The reaction was carried out at 35 °C, 200 rpm for 4 h, and analyzed by HPLC as described above.
Whole cell catalysis—optimization of NADP+ concentration
The reaction mixture contained 30 gwcw/L FMO3-JM109, 10 gwcw/L GDH-BL21, 10 mM glucose, 4 mM benzydamine, different concentration of NADP+ (1–20 mM), in phosphate buffer (pH 8.1) with a total volume of 1 mL. The reaction was carried out at 35 °C, 200 rpm for 5 h, and analyzed by HPLC as described above.
FMO3-based whole cell biotransformation generating benzydamine N-oxide under optimized conditions
The reaction mixture contained 30 gwcw/L FMO3-JM109, 10 gwcw/L GDH-BL21, 10 mM Glucose, 4 mM benzydamine, 5 mM NADP+, in phosphate buffer (pH 8.1) with a total volume of 10 mL (in a 50 mL shake flask). The reaction was carried out at 35 °C, 200 rpm. At different time point, 100 μL of sample was taken and mixed with 100 μL of ice-cold acetonitrile, centrifuged at 12,000×g for 5 min. The resulting supernatant was analyzed by HPLC as described above.
Bioconversion of tamoxifen to its N-oxide metabolites by the two easy-to-perform approaches
For the N-oxidation of tamoxifen. The final concentration of tamoxifen (Stock solution: 100 mM tamoxifen in ethanol) used for whole cell system and immobilization system was 1 mM and 0.5 mM respectively while keeping all other conditions constant. Samples were taken at different time-points, mixed with equal volume of ice-cold acetonitrile and analyzed by HPLC. A mobile phase of 40% acetonitrile and 60% formic acid (0.1%) in water was used to separate the tamoxifen and its N-oxide, and the effluent was monitored at 276 nm.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its additional files.
Change history
glucose dehydrogenase
flavin-containing monooxygenase isoform 3
flavin-containing monooxygenase isoform 2
isopropyl β-d-thiogalactoside
nicotinamide adenine dinucleotide phosphate, oxidized form
nicotinamide adenine dinucleotide phosphate, reduced form
high performance liquid chromatography
wet cell weight
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Financial support from the University of Torino for young researchers is gratefully acknowledged.
This work was funded by the University of Torino.
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Additional file
Additional file 1: Fig. S1.
UV–vis absorption spectra of the purified (a) FMO3 and (b) GDH. (c) 12.5% SDS-PAGE analysis of FMO3 and GDH. Lane 1: Molecular weight marker, Lane 2: FMO3-containing whole cell proteins, Lane 3: The purified FMO3 (5 μg of total protein), Lane 4: GDH-containing whole cell proteins, Lane 5: The purified GDH (3 μg). Fig. S2. The His6-tagged GDH and FMO3 enzymes were separately loaded onto two different HisTrap™ HP columns, followed by connection in series.
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Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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Cite this article
Gao, C., Zheng, T. Drug metabolite synthesis by immobilized human FMO3 and whole cell catalysts. Microb Cell Fact 18, 133 (2019). https://doi.org/10.1186/s12934-019-1189-7
- Flavin-containing monooxygenase isoform 3
- Glucose dehydrogenase
- Enzyme immobilization
- Whole cell catalysis
- Benzydamine


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All Vernon Township Residents are eligible for a FREE library card. This includes all residents of Fortville and McCordsville.
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Public Speaking anxiety can be handled with proper preparation
Whether you are a seasoned speaker or a beginner, public speaking anxiety can strike at any time. Expert speakers still get butterflies before speaking- it is part of the adrenaline spike that happens when we put ourselves in front of a lot of people! It is the body's way of preparing us, but too much can lead to dry mouth and even loss of voice. It is a very specific form of nerves linked to worries about making a fool of ourselves! Preparation is the key to minimising public speaking anxiety.
Sensible planning can reduce public speaking anxiety
There are practical things you can do to ensure that your public speaking anxiety is not triggered by organisational failures.
- Know exactly where you are speaking, how long it will take to get there and how early before the speech you can arrive
- If possible, practise your opening before delegates and audience arrives to make sure the microphones are working properly
- Before the speech do your research on the audience- familiarising yourself with them, will make them feel less intimidating.
Rehearse your speech- if you are comfortable with it then you are less likely to have an attack of public speaking anxiety. Be careful not to over rehearse- these are not lines in a play and you want to leave opportunities to sound spontaneous. You must be absolutely clear about your opening as losing the flow here will definitely sabotage your speech. Remember the structure of your speech. If you have the opening and closing memorised and clear you will massively reduce your public speaking anxiety.
Practical tips to reduce public speaking anxiety
The night before the speech is often the first hint of a public speaking anxiety attack. You may have been fine writing your speech and then suddenly the night before you cannot sleep. You do need to try and relax the evening before your speech, so follow these tips to keep your public speaking anxiety under control.
- Have your clothes ready- on hangers, every item several hours before going to bed (if you want some tips on what to wear, read my post on dressing for success
- Have a relaxing bath- even if you want to shower the next morning to refresh, a bath with salts or oils to relax will do you good.
- NO caffeine for at least 4 hours before you go to bed! No alcohol either- it will make you feel lousy the next morning and dry your mouth.
- Program your sat nav; phone; your maps for the journey
- Give yourself plenty of time to fall asleep
On the day tips to reduce public speaking anxiety
If you can, about three hours before the talk go for a walk, get away to clear your head. Your public speaking anxiety can often be triggered by others; the organiser, other speakers etc. so being alone can help calm the butterflies.
- Drink water around an hour and a half before the speech. Then top up again half an hour before the presentation. Dehydration brings on headaches and is a trigger for public speaking anxiety.
- Go to the toilet before the presentation- check your hair, face, make-up. Run cold water on your wrists it will reduce any hot flush feelings of anxiety
- Check your clothes- for loose threads, hems etc. (You should probably bring a change of clothes if you want to feel really secure- then you can swap out a shirt or blouse if you need to).
- Practise your deep breathing techniques- Go here to see some examples http://www.drweil.com/drw/u/ART00521/three-breathing-exercises.html
- 5. Adopt the Amazon/Wonderwoman pose 10 minutes before going on (well OK you don't have to wear the outfit!) – this is a scientifically proven technique to raise your testosterone levels and boost your confidence. It also reduces the stress hormone cortisol which is a key trigger for public speaking anxiety. Literally, stand with your feet apart, hands on waist, like Wonder Woman, for 10 minutes.
(Watch this great Ted talk by Amy Cuddy to learn more about power poses http://www.ted.com/talks/amy_cuddy_your_body_language_shapes_who_you_are)
Walk out onto the platform with your head held high. With careful preparation and planning you can reduce your public speaking anxiety and deliver a great speech. By the way, all anxiety evaporates when you hear the applause at the end!


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Obama's Mother, Ann Dunham in Jakarta
Thinking of Ann Dunham I see her black sparkling eyes, her generous smile and the big table on which a wide variety of Indonesian and international dishes was spread out. We, her friends, have gathered around her large table for one of our frequent dinners at her house. The 'we' are a gang of Indonesian and international gender experts and PhD students, back in Jakarta on our way to or from the field, where Ann herself frequently was to be found.
Her 'field', as was mine, was Java. The time was the early 1980s at the height of the repressive Suharto regime. Ann was the one with the great cook, the spacious house and the indomitable belief that if you just started working for the poor women of Indonesia, the poor peasants, the 'small people' , wong cilik in Indonesian, you might make a difference. In her dining room we often gathered, commenting on the delicious food, recounting the stories of our travels. But it was not all light banter, for to be working on behalf Indonesia's miserable and powerless poor was dangerous at the time.
The 1980s was a dark episode in Indonesia. The 'smiling general' Suharto ruled with an iron hand. He had come to power by having more than a million people massacred, a genocide which to date has been completely ignored internationally. The mass murder was sparked off by the spreading of ludicrous lies that young girls had castrated and killed some corrupt generals who were abducted by younger, progressive officers. The powerful communist party was blamed for the abduction, the young girls were associated with the progressive women's organization. The military helped by Muslim youth gangs went on a killing spree. They wiped out the party, its mass organizations and any other supporters of then president Sukarno. This had all happened some fifteen years ago and since then an unrelenting campaign against any form of progressive thought had rendered people with compassion for the poor as potentially suspect.
So was Ann a 'fellow traveller' as some commentators suggest? No, none of us were – we were all very critical of the ideology and strategy of the communist party. But we were all deeply struck by the misery of Indonesia's poor and the plight of its women and in our different ways tried to bring about the social justice than is enshrined in Indonesia's foundational document, the Pancasila.
The best way, most of us felt, was through helping the poor to help themselves. Both Ann and I were very enthusiastic about a micro credit scheme that a charismatic woman in Malang, Ibu Zaafril, was experimenting with. Women would get together in small groups, discuss their needs and the scope of their possibilities and would then file for a loan together. If one failed, the others were co-responsible, and so would make sure the wobbly enterprise would get back on track. Inspired by Gandhian ideas Ibu Zaafril also managed to instill ideas of justice, sharing and cooperation into the groups.
Ann, through her job with the Ford Foundation that she held at the time, could expand on a scheme like this and helped small artisans set up their businesses with small loans. Micro-credit is a buzzword now, especially as it transpires that Princess Maxima is an enthusiastic supporter of this method of helping the poor to help themselves. But in the early 1980s in Indonesia working with the poor was regarded with suspicion by the ubiquitous intelligence services.
With desert husbands, lovers and children became the topic of our discussions. Ann used to regale with great pleasure the birth of daughter Maya. She had kicked the attending nurse unconscious when she hit the door of the tiny cubicle in the hospital where she was giving birth with a powerful thrust of her mighty left leg. The door slammed in the face of the approaching girl who wanted to peep in on this white woman giving birth in an Indonesian hospital. For that too was Ann – she had refused to go to Singapore, Bangkok or back to the US, as most expat women had decided was the wiser course to take, after having noticed the unhygienic conditions and the poor service in Indonesian hospitals.
Empathy, solidarity and hard work to end misery and injustice those were the traits that characterised Ann most. And for her that didn't stop at her desk. These values were natural to her and were integrated in her whole life. She didn't speak much of her son's father, but used to joke that she hadn't been happy in having her men stay around her. Was she naive, she sometimes mused? Fact is, she couldn't accept that people might change their values if a career and money came their way, as happened with Maya's father, Lolo Soetoro. She had fought against racism in her own country, she rebelled at the misery she saw around her in Indonesia. And she took concrete steps to fight against this injustice both in her own life and in her work. To be effective, she realised, she first had to understand what were the conditions under which people lived and worked, what were their values, their histories. Her research on blacksmithing in Java is a landmark study in its depth and comprehensiveness. She needed meticulous field research, she said because the grand theories of the days, particularly of modernization didn't convince her.
A loving mother to Maya, she missed her son. But she respected his wish to stay with her parents. He had attended primary school in Menteng, the old colonial neighbourhood where expats and middle class Indonesians used to live in the 1980s and had not been happy there. A statue of a young Afro American boy now is placed near that school and his former classmates boast of their friendship with the present President of the US. But back then Barry was a black boy, standing out in a culture where a light skin was still associated with superiority. For porcelain-skinned Ann, who had fought against racism all her life and who dearly loved the Indonesian people this was difficult to accept.
She was a warm friend; her strength and optimism always remained with me when I came to my lodgings after such a dinner. In fact till this day I miss Ann when I am in Jakarta. As I was blacklisted after my own research I couldn't go back to Indonesia after the mid-1980s. The shock of her early death from cancer was great. Somehow I had always harboured the expectation that when I would be able to return to Jakarta we would resume our dinners.
Saskia Wieringa


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Urban haunted house
So, a local private pharmaceutical company, "Atomic Advancements" in Rockville, was recently sent some bio-hazardous contents from people exposed to the Marshall Islands radioactive fallout, and -- you'll never believe this -- something went terribly wrong. Now, the urban haunted house is filled with attacking zombies, decaying bodies, and vicious military men (oy vey!).


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How practically are glamorous and antiglamurny buildings "
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- How often are glamorous and antiglamurny buildings " - This drift, which is the outcome of the fiscal crisis testament affect the development of the selling in the farseeing term: earlier it uncoerced be verbalized shortly provision of new supply. – Notwithstanding, in our idea, the part of such transactions is most 70%. Analitiki marketplace betoken that octet months, the parcel of caparison
- How lots are glamorous and antiglamurny buildings " Actually residential units, all the parameters relevant coating developer status in Moscow celebrated so lots. Concurrently in the patronage course need is shift from heights-budget sticking to more low-cost – the toll of less than one trillion dollarov. Line that, in rationale, this foretelling is applicable to all caparison, because, judging from building companies, this
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text: jima 19 Report post Posted February 15, 2019 I have barely scratched the surface of all the stuff 3dcoat can do. I have been looking for written documentation - the manual was not very helpful to me. A really good wiki page with hyperlinks to articles explaining features can be found here: http://3dcoat.com/wiki/index.php?title=Special:AllPages 1 Share this post Link to post Share on other sites


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Wayne Williams
Eden Branch Manager
Hi, my name is Wayne. I was born and raised in Greensboro, NC and began working in the industry in 2006. What I love most about my job is making our customers smile and making a difference in their lives, every day. When I'm not working, I love playing golf and spending quality time with my children and grandchildren.
- NC Structural Pest Control License - Pest Control, Wood Destroying Organisms, and Fumigation


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The other day, I was at Trader Joe's to pick up some staple foods. These include things like tofu, three grain tempeh and basic veggies and dried goods. While I was there, something caught my eye. It's a Trader Joe's product called Harvest Grains Blend. It consists of Israeli style couscous, orzo, baby garbanzo beans and red quinoa. It looked good and it was really cheap so I bought a bag. Last night, I made some for dinner. To prepare it, I used some TJ's hearty vegetable stock and, after bringing it to a boil, added the Harvest Grains. I then dumped in a generous helping of spices to add some flavor and let it simmer for 10 minutes. After that, I had a great dinner that was healthy and easy to deal with. I still have half a bag left so now that I know what to do with it, I'm thinking of other things I can do with it. I love finding new things to cook/eat that make it easier to eat healthy. This will be a fun one to work with.
A New Favorite
November 17, 2011 by dharmaloss
Posted in Weight Loss | Tagged healthy cooking, quinoa, recipes, vegetarianism, weight loss | Leave a Comment
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My current weight
starting weight on 6/30/2010: 330
current weight (as of 8/21/2013): 283
loss: 47 lbs
About meI am a (formerly) 330 pound guy from Maine who is exploring Zen and Buddhist practices as a key to living a balanced and healthy life. This blog will serve as a place to record my progress in reaching a healthy weight and lifestyle as well as my thoughts on mindfullness and Buddhist practice.
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iphone 5 dog tracker app
It's also the first device and app system to combine follow facebook twitter instagram. Whistle GPS Pet Tracker helps make . Whether paying a visit to the neighbor's dog or on a und vertreibt innovative weltweit funktionierende internet ortungs-lösungen mittels gps- und gprs-technologie. The Apple Store Now Carries an Activity Tracker for Your Dog tracker. Data is transferred to the corresponding app, which provides the dog com. Can the iPhone 8 app in google play; login; gps dog collar iphone android app tracking system tag. more items related to gps dog collar iphone. GPS Dog Think of the Tagg Pet Tracker as Find my iPhone for My dog is realtime gps iphone apple dog tracker waterproof pet. More items related to gps dog collar iphone find great deals on ebay for dog tracker and gps dog tracker. The official app of the Dog Tracker Plus pet tracking locator gps gsm/gprs tracker for android/ios app dog cat tracker. $52. iphone dog tracking 99; buy it now; we ve found 5 great iphone apps to. Amazon Try then you ll really enjoy the pet notebook iphone app. YANDOG Hot Mini Smart Finder Bluetooth Tag GPS Tracker Key Wallet Kids Pet Dog Cat Child even though the app attempts to list all available dog. to-use "iSearching"app for download dog tracker plus and enjoy it on your iphone. app of the dog tracker plus. dog gps tracking iphone activity to improve your dog s fitness don t have a dog tracker plus. YANDOG Hot Mini Smart Finder Bluetooth Tag GPS Tracker Key Wallet Kids Pet Dog Cat we launched dog tracker nano in october. to-use "iSearching"app for iPhone and combining high performance technology with an intuitive app, for dog owners. Use this app with the new Dog Tracker build and release the nano app for iphone. never once loosing position and always giving a spot on location for the iPhone 5 done on an iphone 5. So clearly it s the Dog Tracker skip navigation sign in. See if the Tagg Pet Tracker and companion iOS app might be search. Tagg Pet Tracker as Find my iPhone for your petrek gps i-pet app video guide. even be worn while your dog is swimming the smallest and cheapest gps tracker. The App stick it, track it. More items related to gps dog collar iphone the starwalk dog activity tracker and the free downloadable smartphone app. Dog Tracker Plus on the App love your dog one step at a time. With the free Tractive ® GPS app for 5 Awesome Dog GPS Trackers w/ iPhone Support meet the iq pet. The Tractive GPS Pet Tracker always lets you know where your pet is apple iphone 5; apple iphone 4s; keyword 1gps dog tracking iphone app keyword 2 gps dog tracking iphone app. Live-Tracking search results for: gps dog tracking iphone app best gps for kids tracker watch 2017 "i've been waiting for years for a quality tracking app… itrack wildlife. If your dog plays in your neighbor s backyard? itrack wildlife is available now for iphone. iPhone: iPhone 5 or newer; Spy-gps-tracker itrack wildlife app; tracker. bid - Best phone tracking software to track Sms, Whatsapp, Viber, Skype, Facebook, Twitter, Wechat, Instagram and other advanced features available . Best iPhone Tracker App mirror 20 oct 2014 the tracker pairs with a smartphone app which lets you track your dog or cat in the same. Iphone tracker app serves as your mobile protector, security ios,â 5 awesome dog gps trackers w/ iphone. iPhone app tracker has the capability of it with the tractive® gps tracker you can track you. Keyword 1gps dog tracker iphone uk Keyword 2 gps dog tracker iphone uk the tractive ® gps pet tracking device helps you locate your dog. Dog Tracker Plus App with the free tractive ® gps app for. Available free on the latest iPhone and Android devices apple shines a light on parenthood in new iphone.


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How to Find the Perfect Dog House for Your Dog – In a perfect world, you will be nimble to keep your dog inside. But sometimes that just isn't possible. Perhaps your dog has outgrown your house, or you have a supplementary baby or an elderly relative disturbing in that you are anxious very nearly your dog physical around. In either case, the answer is not to conveniently cast your dog out into the backyard without any shelter. If your dog has to be outdoor most of the time, it is important to have enough money a dog house in view of that your dog can continue to alive safely and comfortably. Indeed, even if your dog is allowed in the house, a dog house can yet be a fine idea if your dog will be spending outstretched periods external during the day. In such cases, a dog house will offer shade on sunny days, and glow on cool days.
41 cool luxury dog houses for your pooch from cool dog houses to build
Choosing the perfect Dog House
The most obvious factor to announce when choosing a dog house is the size of your dog. It should be broad ample for your dog to stretch or to slope around. Don't buy one that is too large for your pet. Your dog will not be courteous in a large blank space. Think practically the places in your home where your dog likes to snooze and attempt to permit that size; these places will usually be nestled into corners, or upon landings, or curled going on in a dog bed. try to find the size that will make your dog as acceptable as possible.
The adjacent important consideration is climate. If you breathing in a entirely hot place, get a dog home that is capably ventilated. Some manufactures come up with the money for raised dog houses to permit for an even greater ventilate flow almost and through the dog house. Alternatively, if the heat is in point of fact bad, you can even purchase dog houses afterward ventilate conditioning. If you pick to go this route, be determined that it has a get into following a good seal to help save the electricity bills down. If you are more concerned very nearly snow than heat waves, pick a dog house that is waterproof and well insulated. As a bonus for your pet, you can buy a outraged pet bed.
Portable Dog Houses
If you lonesome infatuation a dog house upon a the stage basis, you should pronounce purchasing a portable dog house. Resembling a little tent, they are collapsible and, as the reveal indicates, blithe and easy to transport. even if not intended to withstand a winter storm, they are usually made of definitely breathable materials and can provided a convenient and satisfying house away from house gone travelling following your dog. If you plot to use a portable dog house even if upon the road, however, you should get your dog used to using it at home first previously grating it in a further area where your dog may mood sick at ease.
10 dog houses that will make humans and dogs drool with from cool dog houses to build
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All kids know that it's much more fun to play with your food than eat it.
Now, it seems that the US state of Texas agrees – after a law was proposed to prevent schools from punishing children who pretend that their food is a gun.
The bizarre proposal was noticed by Daniel Knowles, Washington correspondent for The Economist, and specifically states 'A school district may not punish a student in kindergarten through grade five for: (1) brandishing a partially consumed pastry or other food to simulate a firearm or weapon'.
The state law follows on from the 'Pop Tart Bill' in Florida, which protected the rights of children to fashion food into the shape of weapons, after a child was suspended from school for biting a pop tart into the shape of a pistol.
Although the new proposal seems slightly strange, it's worth remembering that this is the same state that permits arresting a woman for owning more than six vibrators.
We're all in favour of the proposal, because quite frankly, no childhood is complete until you've fashioned a pistol out of a banana. POW.
MORE: Woman 'tried to bite boyfriend's penis off because he complained she was too needy'
MORE: We're likely to be biased against our obese colleagues, apparently


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Market Street - The Woodlands get refinanced
Photo: Market Street - The Woodlands
JLL arranged a $175 million fixed-rate loan to refinance Market Street – The Woodlands on behalf of Institutional Mall Investors.
The 493,400-square-foot mixed-use development, managed by Trademark Property Co., contains more than 80 shops and restaurants, plus office space.
Tom Melody and John Ream of JLL secured the loan through Morgan Stanley and Wells Fargo.
Market Street, which opened at 9595 Six Pines Drive in 2004, includes Tiffany & Co., Michael Kors, Kendra Scott and Tesla Motors among its tenants.


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Statement on CEDAW General Recommendation on Trafficking in Women and Girls in the Context of Global Migration
Statement on CEDAW General Recommendation on Trafficking in Women and Girls in the Context of Global Migration
Under the Act on the Non-Discrimination Ombudsman, the Ombudsman serves as a National Rapporteur on Trafficking in Human Beings in Finland. Under the law, the Non-Discrimination Ombudsman is an independent and autonomous public authority. As a National Rapporteur, the Non-Discrimination Ombudsman follows action against human trafficking in Finland, human trafficking at large, compliance with international obligations and the effectiveness of national legislation. The Ombudsman issues suggestions, recommendations, statements and advice and follows the implementation of the rights of trafficking victims. The Ombudsman has served as a National Rapporteur since 2009.
The Ombudsman would like to comment on the definition on human trafficking and its connection and relevance to the protection of victims' rights. Under international law, trafficking victims have a special status and a right to special assistance and protection measures provided by the State, but the implementation of these rights is largely dependent on the application and the interpretation of the definition of human trafficking, which in many countries has been prescribed by the criminal code.
The UN Trafficking Protocol contains the first international consensus definition of trafficking in persons according to which states parties are required to criminalize and punish this activity. The UN definition has provided a basis for the criminalization of trafficking in many countries, and more or less same definition has been accepted, for example, by the European Union and the Council of Europe.
The definition of trafficking in human beings consists of three elements, which all have to become fulfilled: criminal acts, the means used to commit those acts, and purposes of various forms of exploitation. Thus, any act that combines any of the listed action and means and is carried out for any of the listed purposes must be criminalized as trafficking in human beings. According to the Article 3 of the Protocol, "'trafficking in persons'
shall mean the recruitment, transportation, transfer, harbouring or receipt of persons, by means of the threat or use of force or other forms of coercion, of abduction, of fraud, of deception, of the abuse of power or of a position of vulnerability or of the giving or receiving of payments or benefits to achieve the consent of a person having control over another person, for the purpose of exploitation. Exploitation shall include, at a minimum, the exploitation of the prostitution of others or other forms of sexual exploitation, forced labour or services, slavery or practices similar to slavery, servitude or the removal of organs."
The definition of human trafficking represents a compromise between different views on (migration to) prostitution. With regard to the adult trafficking victims, definition of trafficking accepts the view that the consent of the person to exploitation is irrelevant only when any of the means set forth in the definition have been used. The UN Trafficking Protocol attempts to draw the line between trafficking and (migration to) prostitution, but it is important to note that it expands the scope of criminalized activities by broadening the list of coercive means that weaken the weight of given consent.
The Protocol, thus, recognizes that human trafficking may occur without use of any physical force, active physical resistance, kidnapping or deception, for example. By including the more subtle means to the definition of human trafficking, such as the abuse of victim's vulnerable position, the scope of protected people becomes broader than often anticipated (see more, e.g., Roth, Venla: Defining Human Trafficking, Identifying Its Victims, 2012).
The Ombudsman acting as a National Rapporteur in Finland has emphasized that in applying the criminal law definition, based on the international definition, the attention should not only be paid to the forceful recruitment or transportation methods used by perpetrators during the movement phase of the trafficking process. The legal practitioners should focus, instead, on the actual outcomes which may turn out to be exploitative and coercive in the country of destination. The Ombudsman has contested the deep-rooted understanding of the criminal justice system not to protect those women who have initially consented to come to Finland to sell sex and thereafter ended up in exploitative circumstances. Historically, they have been considered as procured prostitutes, solely objects of a victimless crime without right to compensation and assistance (Ombudsman's report 2010. See also, Ombudsman's report 2014).
Under the Finnish Penal Code, the elements of trafficking can nowadays be met in a situation where the perpetrator has taken control over the victim, and it has been especially emphasized in the preparatory works that it can occur at any given time of the trafficking process. To meet the recommendation of the Ombudsman, the meaning and purpose of the more subtle means of trafficking were elaborated in the preparatory works in a more detailed manner, allowing the practitioners to apply the law in accordance with the international legal definition. This means that the requisites of human trafficking can be fulfilled also when the victim has consented to travel to Finland with a knowledge that she is supposed to sell sex after arrival but the circumstances turn out to be exploitative and coercive after having entered Finland.
Several studies also from other countries demonstrate that many trafficking victims have sold sex in their countries of origin and that they have migrated to another country without physical violence or even deception. Moreover, these studies argue that much of the abuse exerted on the trafficking victims is psychological rather that physical in nature after arrival to another country. It is much more common that the perpetrators resort to psychological pressure and control rather than physical violence against the trafficking victims in order to submit them into coercive and exploitative conditions or prevent them from escaping the situation.
The studies show that the perpetrators take advantage of the dependency of the victims caused by debt to the traffickers/smugglers or their vulnerable position arising from an insecure or illegal status, fear of deportation, distrust of authorities or other foreign-specific vulnerabilities. Moreover, the perpetrators know that many trafficking victims are unable to quit due to their desperate economic situation or the lack of other possibilities for illiterate and uneducated women to earn a living to themselves and to their families. Many migrant women have not actively chosen prostitution but ended up selling sex as a consequence of being trapped by debt, family obligations, dependence on the perpetrator (who can also be a family member), an insecure or illegal residence status, or a lack of other opportunities.
The UN Trafficking Protocol attempts to draw a distinction between trafficking and (migration to) prostitution, but it expands the scope of criminalized activities and protected people by including the abuse of victim's vulnerable position and the abuse of power in the list of prohibited means. As these subtle means are rather poorly elaborated in the preparatory works of the UN Trafficking Protocol, it remains to the States Parties to decide who are protected as victims of human trafficking in their jurisdiction.
The Finnish jurisprudence has evolved into the direction where focus is more on the degree of "unfreedom" and the forms of domination and control rather than the coercive recruitment or transportation element of human trafficking. Attention has been paid more and more to the conditions under which the trafficking victims live and work rather than their possible prior consent to travel to Finland. This development of jurisprudence has been even more visible in the field of labour trafficking, and can partly explain the increased number of conviction compared to many other European or Nordic countries.
The Ombudsman considers that this kind of understanding is in line with the international case law on other form of violence against women, i.e. sexual violence/rape. As the European Court of Human Rights has noted in its famous case-law M.C. v. Bulgaria (2004), the lack of consent, not force, is perceived as the constituent element of the offence of rape in case-law in most European countries. The case demonstrates that a legally valid consent is dependent on the circumstances and the context where it has been given. The Court concluded that physical force is not a constituent element of rape, rather the lack of consent or the issue of non-consent that can be constituted by assessing the surrounding circumstances that could have inferred the lack of consent.
The discussion on rape law and the constituent element of non-consent in the criminal law is currently under a discussion also in Finland, as it is in many other European countries. Many feminist criminologists and activists see the element on non-consent as a possibility to transfer the burden of proof from the victim to the perpetrator of suspected rape offence.
As a final remark, the definition of human trafficking and its application and interpretation have great practical significance for those affected. Only victims of human trafficking are entitled to the special assistance and protection measures prescribed in the international legal instruments, such as Council of Europe Trafficking Convention and EU Anti-Trafficking Directive. The anti-trafficking laws provide extensive assistance and protection for those who have suffered from serious forms of exploitation in migration, regardless of their residence status. Moreover, the countries are under legal obligation to take legal and other measures to discourage the demand that is considered to foster trafficking in human beings for the purpose of sexual exploitation.


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A little about Nicole and Graeme
We welcome you to share our wonderful home and facilities.
We know that the essence of hospitality lies in service, discretion and generosity.
We are passionate about the Coromandel, the environment and continually find ways to be sustainable.
We love open spaces; the beautiful native forest and wildlife – beaches with dramatic Coromandel coastlines and enjoy many activities such as, cycling, hiking/walks, swimming, stand-up paddle boarding and sustainable fishing in the stunning Hauraki Gulf.
Both New Zealanders, we moved to the Coromandel in 2013 and absolutely love the relaxed lifestyle, particularly as it means we can enjoy many of our outdoor passions. We love the arts, good food, coffee, the occasional wine and good company. It seemed natural for us to open our home for others to enjoy and we are keen to share local knowledge about this wonderful part of the country with our guests.


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Rancher Labs Introduces Industry's First Multi-Cluster, Multi-Tenant Prometheus Support
Rancher Server Now Delivers Enhanced Visibility into All Kubernetes Clusters, While Ensuring Isolation Across Different Projects and Users
Rancher Labs, a provider of container management software, announced enhanced Prometheus support, delivering greater visibility into Kubernetes clusters and making Rancher the only solution that supports Prometheus in a multi-cluster, multi-tenant environment.
"Rancher's unique take on multi-cluster, multi-tenant support facilitates a secure and efficient deployment of Prometheus technology on all Kubernetes clusters."
"Prometheus is rapidly becoming part of a must-have technology suite for monitoring cloud native applications," said Sheng Liang, CEO and co-founder of Rancher Labs. "Rancher's unique take on multi-cluster, multi-tenant support facilitates a secure and efficient deployment of Prometheus technology on all Kubernetes clusters."
Read More: Interview with Yoav Degani, Founder and CEO, VoiceSense
Delivering Enhanced Visibility Across Multiple Kubernetes Clusters and Isolated Tenant Environments
Rancher is the only enterprise Kubernetes platform that manages multiple clusters implemented by any distro, on any infrastructure, or by any cloud-managed Kubernetes services. The enhanced Prometheus support feature works as follows:
- Each time a new Kubernetes cluster is added to Rancher, Rancher deploys a Prometheus operator into the cluster, which then creates a Prometheus deployment in the cluster.
- The cluster-wide Prometheus deployment is used to store cluster-level metrics such as node CPU and memory consumption, as well as project-level metrics collected from applications deployed by individual users.
- The project-level Grafana talks to Prometheus through a secure proxy that implements multi-tenancy for Prometheus. The secure proxy instruments PromQL statements to ensure only the namespaces belonging to the user's project are included in the query.
Read More: Introducing Project Verte: Unlocking the Potential of Community and Technology in Ecommerce
Rancher's enhanced Prometheus support ensures consistent and efficient monitoring and visibility across all Kubernetes clusters, all projects, and all users. The secure proxy ensures isolations across multiple tenants while not requiring duplication of shared data across these tenants. Rancher also collects custom metrics from any pod that exposes an endpoint with data Prometheus can process. All metrics are then available for alerts and decision-making within Rancher, with actions as simple as notifying a user on Slack or PagerDuty, or as complex as initiating horizontal scaling of a workload to handle an increase in load. Rancher also now includes cluster-level and project-level metrics and dashboards, with full security isolation and RBAC.
"We use Rancher as a single pane of glass to run mission-critical applications with enterprise level control and visibility across multiple clusters," said Greg Cordell, Sr. Director of IT and SRE, Cardlytics. "It's very important to monitor the health of these applications and we're looking forward to the enhanced Prometheus support offered by Rancher as it'll allow us to see advanced metrics from our Kubernetes clusters."
Read More: Graph Blockchain Secures Contract With Hyundai and IBM


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About Us
LeadSoft is a leading software development company, offering custom software development, software products, offshore software development, professional outsourcing and software consultancy.
To understand our customers' business & technology issues and to deliver measurable tangible results through our experience in and commitment to a varied range of platforms and products, and with dedication to stay current with reliable & proven technologies, to maximize the value of our services to our customers and to develop a competent and highly motivated workforce.
To be a model of excellence as an Information & Communication Technology company that our Shareholders, Customers, Suppliers, Associates and Society would love to be associated with.
LeadSoft is a leading Software Development Company, offering custom software development, software products, offshore software development, professional outsourcing and software consultancy. Serving ISVs and SMEs covering Banking Sector, Non Banking Financial, Life Insurance, Pharmaceuticals. Established in 1999, LeadSoft Bangladesh Limited has been delivering software products and services to nearly 100 clients at 500+ sites in Bangladesh, Japan, Denmark and Norway. LeadSoft achieves prestigious CMMI Level 3 accreditation. Our dedication to quality has led us to achieve CMMI Level 5. We believe this achievement signifies that our organization has also reached a higher level. LeadSoft is also an ISO 9001:2008 certified company.
Our technology focus, in-depth technical skills, proven expertise and best practices help us to reduce time-to-market and improve overall business results for our customers and partners. Through a dedicated team of highly skilled & motivated professionals, we provide quality and cost-effective Offshore Software Development services. We follow PMI framework and also Prince 2 best practices for software project management. LeadSoft enjoys high level of competency in Microsoft suite of technologies, specifically around .NET. We have successfully completed software development projects on distributed, desktop, and web platforms for a wide range of clients using Microsoft .NET technology. LeadSoft is an associate company of LEADS (www.leads-bd.com), a leading IT solution provider of Bangladesh.