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Welcome to America! …The Land of Unintended Consequences. We have the greatest of intentions with an alarming degree of shortsightedness.
Below are 9 ugly examples of unintended consequences. Unintended consequences are outcomes that are not the ones intended by these well-meaning actions. Leave your party affiliation at the door.
1) We create a minimum wage to help the working poor.
The unintended consequence is that the lowest skilled workers become unemployed. Under minimum wage laws it is illegal to hire the lowest skilled workers for what their labor is worth to an employer. Shame on us.
Solution: Eliminate minimum wage laws.
2) We offer welfare for those in need of assistance.
The unintended consequence is that many on welfare find it more advantageous to stay on welfare rather than working to support themselves. Our efforts to help those in need have led to the subsidization of bad behavior. Sheesh people.
Solution: Eliminate government welfare. Welfare should be privately managed in a free society. As organizations reliant upon private, freely offered donations, privately managed welfare is accountable to donors and has an incentive to reduce fraud and abuse.
3) We pass Affirmative Action laws to help minorities get into the workforce.
The unintended consequence is that the low skilled minorities have a harder time getting hired. Low skilled minorities come with higher legal risks when compared to non-minorities of similar skills. For example, the financial and legal risk for a small business owner to attempt to fire a poor performing minority is greater than the risks associated with a non-minority. Hence, these protected minority groups have a harder time finding employment. Way to help out, people.
Solution: Eliminate all Affirmative Action laws.
4) We pass bans on guns to reduce gun violence.
The unintended consequence is that law abiding citizens are less safe. Law abiding citizens that follow the rules make themselves easier victims for non-law abiding citizens with guns. We protect ourselves
and our children "No Guns Allowed" signs instead of guns. Way to go.
Solution: Preserve the Second Amendment.
5) We criminalize drugs to make people safer.
The unintended consequence is that people, especially low income minorities, are less safe. Because drugs are illegal, violence, instead of rules of commerce, regulates the industry. Also, millions of people, especially (and unfairly) low income minorities, are jailed for drug offenses that amount to a non-violent crime that did not infringe upon the rights of anyone else.
Solution: Legalize drugs. All drugs. People own their own bodies. We should not lock people up in cages for behavior that is destructive to themselves and nobody else. Are we free people or not?
6) We set up rent controls to make housing affordable.
The unintended consequence is housing shortages. Renters that would have downsized their rental apartments will instead choose to squat in rent-controlled apartments because there is no economic incentive to align housing square footage with their reduced needs. This leads to housing shortages. In addition, apartment owners often abandon their severely dis-repaired buildings. This is because the artificially low rental income does not align with expenses required to maintain buildings. The Rent is Too Damn High Party consists of one indignant (but well intended) boob.
Solution: Eliminate Rent controls.
7) We promote home ownership by keeping interest rates down.
The unintended consequence is that people lose their homes in a housing crash. In a free market interest rate environment, rates would fluctuate naturally based on market fluctuations. When interest rates rise, the prices of homes will decline as the demand for homes declines. However, when interest rates are forced artificially low, home buyers can magically afford more than they could under free market conditions. As a result, home buyers bid up the prices of homes with their cheap credit as their leverage. This incentive for home buyers to over-leverage in debt inflates a housing bubble that eventually has to pop. The salt in the wound is when the people lose their equity – big banks are bailed out. This bailout, of course, is courtesy of these same taxpayers that lost their equity in their homes.
Solution: Eliminate the Federal Reserve. No organization, especially a government arm, should have the power to manipulate interest rates.
8) We create the FDA to keep our food supply safe.
The unintended consequence is that our food supply is less safe. A megacorporation called Monsanto has infiltrated the FDA regulatory and legislative process pushing their corporate agenda over our public food safety concerns. Consumer interests are second to corporate interests. Who wants seconds?
Solution: Eliminate the FDA. Are you telling me that people are incapable of organizing private organizations to certify to quality of food and drugs? These private organizations would be accountable to the people instead of the corporate masters that dominate the FDA.
9) We create the FDA to make safe prescription drugs available to the people.
The unintended consequence is that people are blocked from innovative drugs and
arrested for using "illegal" drugs that treat their symptoms. The FDA drug approval process is built to protect the interests of existing big pharma powerhouses. Creating a new, innovate drug on a low budget is a threat to big pharma's bottom line. Permitting ordinary people to grow a marijuana plant in their back yard to treat glaucoma symptoms is also not compatible with the interests of big pharma. The FDA serves its big pharma corporate masters.
Solution: Eliminate the FDA. See the solution for #8.
How many regulations, laws, and government agencies have been created with the best of intentions but led to distortions that are worse than our original maladies? Giving the government the power to do good is also giving them the power to do bad. Instead of giving the government the power to do good, we should limit their power. The free market is a much more effective means to regulate. We can take any example cited above, remove government interference, and find ourselves collectively in a much better position.
"Power to the people" is not just a catchy line in a Public Enemy song. Power to the People has real, tangible benefits. Giving Power to the Government does not serve the interests of the people.
"Fight the power… Power to the people no delay!"
Knowledge = Power. Peace. Freedom.
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The British brand invites customers to purchase items straight from its London fashion week collection.
In the final stages of preparation, the anticipation of Burberry's A/W 2013 collection builds as fashion's elite wait to see how designer Christopher Bailey will top last season's metallic wonderland collection.
Thankfully, Burberry's customary live stream means you won't have to wait long to see the new fall designs, or get your hands on them – Burberry's new Runway Made to Order service is offering custom-made coats and bags available to purchase straight off the runway until Sunday 3 March 2013.
The new bespoke service is just one of many technological innovations that the British luxury label is incorporating into its' upcoming collection – head over to au.burberry.com for more details.

subset: commoncrawl


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The state will use a carrot-stick approach to keep unseaworthy boats from sinking and polluting Evergreen State waters, under a bipartisan bill signed into law Wednesday by Gov. Jay Inslee.
The bill is principally the work of two islanders, Reps. Drew Hansen, D-Bainbridge Island and Normal Smith, R-Langley. Smith was spurred on by the sinking of a clunker fish boat at Coupeville with pollution that forced the temporary closure of the Penn Cove mussel beds.
"We have raised the bars for buyers and sellers of high-risk vessels, creating clear expectations," Smith said, "and we've lowered the cost of deconstruction to help owners do the right thing at the end of a vessel's life cycle."
The bill would:
— Establish an annual vessel-removal fee of $1 per foot on all commercial vessels required to be listed by the state Department of Revenue;
— Require that marine insurance be held by moorage facilities and boats moored on the premises;
— Require that insurance be held by sellers and buyers of boats over 40 years old and longer than 65 feet;
— Prohibit sale of unseaworthy boats over 40 years of age and over 65 feet.
— Provide exemption from the retail sales and use taxes in cases where vessels are in the process of deconstruction.
"This bill will save jobs and also keep our waterways clean and safe for recreation and industry," Hansen said.
Inslee praised the state Department of Natural Resources for its Derelict Vessel Removal Program, and what he called work on an issue that "galvanized both sides of the aisle."
The state is getting tough with clunkers on the water. Attorney General Bob Ferguson is providing help to local prosecutors and leveling charges, most recently against the owner of a vessel, expelled from its moorage, that sank in the Columbia River below the Tri-Cities.


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Wednesday, September 30, 2009
Pouf a la Cupcake
Oh dear. I've found some more affordable artwork on Etsy for you. That is...if I don't buy them all up first! I stumbled upon Elizabeth Ocean's watercolor prints and it was love at first site as soon as I saw her Chapeau series. Somehow Elizabeth has managed to combine two of my favourite things, food and 18th century fashion, in fantastic watercolor prints that would make a great series in anyone's living room. One of my favourite's has to be Le Chapeau des Langoustines Jumelles, especially since it's a shout-out to the artist's native Boston roots. I'm already envisioning these on my wall in equally fabulous rococo frames! Check them out here.
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Time Sensitive
When you're running short on time and options, you need a carrier that understands on-demand, urgent needs. Our Time Sensitive services provide the specialized capacity you need – when you need it.
Whether it is a last-minute load, a one-time or annual project, an end-of-the-quarter pop-up fleet, disaster relief, or a single shipment with special requirements, we'll find solutions that meet your needs.
The drivers who haul our Time Sensitive freight have a proven track record of success and understand the mission-critical nature of safely hauling highly-sensitive expedited freight.
Our Safety Standards
All employees maintain a high level of safety as reflected in our CSA 2010 Scores and a satisfactory rating with the U.S. Department of Transportation. WTC is also the recipient of the Silver National Safety Award, awarded by Great West Casualty Company. Great West is the premier provider of insurance products and services for truckers and they specialize in insurance products exclusively for the trucking industry. The award recognizes WTC's outstanding achievement in highway traffic safety for the years 2009 and 2010.
The Safety Department in Milton, PA continually monitors and enforces our policies and procedures, ensuring a high level of quality, service, and dependability.
Our Innovative Technologies
Anyone with a fleet of trucks knows that there is a science and art to fuel efficiency. WTC has an entire staff dedicated to increasing the fuel efficiency of the fleet – from adding and adjusting wind deflectors on the trucks, to monitoring tire pressure, the team is constantly measuring changes to fuel economy.
Each truck is equipped with satellite tracking and Electronic Data Interchange (EDI) interfacing technologies.
Satellite based systems allow us to fully monitor, track, and improve the performance of our fleet and efficiency of our client's shipments.
These innovative technologies also allow the home office to communicate with the fleet, in case issues may arise. This gives our drivers and customers peace of mind.
WTC participates in the U.S. Environmental Protection Agency's SmartWay Program. The Program:
- Assesses freight operations
- Calculates fuel consumption and carbon footprint
- Tracks fuel-efficiency and emission reductions annually
Watsontown Trucking Company has received the Program's highest rating


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Apple's Beats brand in April unveiled the Powerbeats Pro, a redesigned wire-free version of its popular fitness-oriented Powerbeats earbuds.
MacBook Pro Updates
Apple Shutting Down iWork.com on July 31 Amid Transition to iCloud
Apple today sent out an email to users registered for its iWork.com beta noting that it will be discontinuing the service as of July 31. The discontinuation comes as Apple places its focus on iCloud, which offers users ways to keep documents in sync among their various iOS devices.
Dear iWork.com user,iWork.com launched as a free beta in January 2009, with Apple eventually intending to offer the service with tools for collaboration on iWork documents for a fee. The service never exited beta, however, and with iCloud offering new options for moving documents around the company has decided to eliminate the program. But while iCloud does offer a number of new features, it does not offer the level of collaboration tools envisioned for iWork.com.
Thanks for participating in the iWork.com public beta.
Last year, we launched iCloud, a service that stores your music, photos, documents, and more and wirelessly pushes them to all your devices. Today, there are already over 40 million documents stored on iCloud by millions of iWork customers. Learn more about iCloud.
With a new way to share iWork documents between your devices using iCloud, the iWork.com public beta service will no longer be available. As of July 31, 2012, you will no longer be able to access your documents on the iWork.com site or view them on the web.
We recommend that you sign in to iWork.com before July 31, 2012, and download all your documents to your computer. For detailed instructions on how to save a copy of your documents on your computer, read this support article at Apple.com.
The iWork team
The elimination of iWork.com will come a month after Apple officially shuts down its MobileMe services. Apple has continued to support MobileMe since the introduction of iCloud last year in order to give users time to move over to the new service. But as of June 30, MobileMe and several of its features such as iDisk, Gallery, and iWeb publishing will no longer be available.
Top Rated Comments(View all)
Obviously since you cannot access iWork beta from Pages, Numbers & keynote after July 31st, sounds like an update to remove iWork and add iCloud will FINALLY be coming!
Not everyone has Lion.
Your customers
Seriously, my MBA is owned by my employer, who does not want to upgrade to Lion. Despite my telling them that it's an easy upgrade (and I'll pay for it) they don't want the possible headache of incompatible software.
If apple would make iCloud work with Snow Leopard, this would be acceptable. I mean, iCloud works with Vista for crying out loud.
It's so stripped down and not very functional compared to google and more or less ms office
Thats fine as long as they get their heads out of their...clouds, and get document handling fixed for iCloud. Great, so I can have my docs in the cloud, if I create them on an iOS device. On my mac, I have to manually upload and download them? Very very very unApple.
If I create a doc in Pages on my mac, it should go to iCloud, unless I tell it not to. It should show up just like if it was created on an iOS device. I'm really not sure why it isn't already like that.
No doubt coming with Mountain Lion.
In Mountain Lion, several apps (Text Edit and Preview) have "iCloud" as an option in the Save As dialog. And when you open Preview you get a window with the images you have saved in iCloud.
July seems about right for the ML release.
Apple shouldn't be held back from moving the operating system forward just because some people don't want to upgrade.
Should Microsoft hold back updating Office or Internet Explorer because some companies don't want to upgrade from Windows 98, NT, or XP?
I'm not asking them to hold back the OS, just asking them to include iCloud in Snow Leopard. I'm all for them bringing out Lion and Mountain Lion and whatever the next big cat is. Like I said, if they can do it for Vista, they can do it for SL.
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Denso VP on developing concepts for tomorrow's car air-con - Q&A
Continuing just-auto's series of interviews with tier one suppliers, Matthew Beecham spoke to Roland Priesemuth, Vice President Business Development, Denso Thermal Europe Centre about the key market drivers for climate control technologies.
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just-auto gives its paid members access to the very best automotive market coverage.
And now there's just-auto plus, our premium membership that gives you exclusive component forecast data, company profiles and extended news coverage - just for premium members.
Today I can offer you 30 days access for $1 - and that includes just-auto plus.
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Wednesday, November 19, 2008
Katie Newman Gifts for Home
I love the Victorian look with the soft pinks and flowers everywhere. Katie Newman Gifts For Home is a website full of the perfect gifts and trinkets. If I had money to spend I would definitely spend a lot of time on this website. Everything is so beautiful. There are gift baskets, wall decor, jewelery, books and so much more. Every woman will love this site and any gift that comes from it. I received this wall hanging and it was only five dollars! It is beautiful and has the saying "Love doesn't make the world go round, Love is what makes the ride worth while." For beautiful gifts that are reasonably priced visit Katie Newman Gifts and Home.
Posted by Sandra at 7:15 PM
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Courteney Cox and Snow Patrol's Johnny McDaid reportedly plan three-day Irish wedding
Courteney Cox could beat her former Friends co-star Jennifer Aniston up the aisle.
The former Friends stars are both currently loved up, with Jennifer announcing her engagement to Justin Theroux in August 2012 and Courteney stepping out with boyfriend Johnny McDaid in December last year.
Speculation about Jennifer's impending nuptials have been rife since Justin popped the question, but now it seems 49-year-old Courteney might get their first.
"Courteney and Johnny are crazily in love. They've moved in together and can't see any reason to wait for a wedding," a source divulged to British magazine Star.
"They believe they're soulmates and this is the next step. It seems they'll tie the knot before Jen!"
Courteney has been married once before to David Arquette, but they finalised their divorce last year.
She's now well and truly moving on, with the source adding that she and Johnny plan to have three days of festivities in Ireland, where the Snow Patrol singer hails from.
"They've been to see venues and agreed on an idyllic castle on the west coast," the insider continued.
"And they've been talking about a date in spring."
Courteney has daughter Coco, nine, with David.
Her daughter, warming to her new flame, has apparently cemented the idea of a wedding for Courteney.
"Coco has met Johnny and she really likes him," the source explained.
"It has convinced Courteney he's right for her. David has also met him and really likes him, too. Courteney gets on well with David's new girlfriend Christina McLarty - who's expecting their child - and it means a lot to her that they can all be friendly."
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The gut microbiota has been proposed as an environmental factor that promotes the progression of metabolic diseases. Here, we investigated how the gut microbiota modulates the global metabolic differences in duodenum, jejunum, ileum, colon, liver, and two white adipose tissue depots obtained from conventionally raised (CONV‐R) and germ‐free (GF) mice using gene expression data and tissue‐specific genome‐scale metabolic models (GEMs). We created a generic mouse metabolic reaction (MMR) GEM, reconstructed 28 tissue‐specific GEMs based on proteomics data, and manually curated GEMs for small intestine, colon, liver, and adipose tissues. We used these functional models to determine the global metabolic differences between CONV‐R and GF mice. Based on gene expression data, we found that the gut microbiota affects the host amino acid (AA) metabolism, which leads to modifications in glutathione metabolism. To validate our predictions, we measured the level of AAs and N‐acetylated AAs in the hepatic portal vein of CONV‐R and GF mice. Finally, we simulated the metabolic differences between the small intestine of the CONV‐R and GF mice accounting for the content of the diet and relative gene expression differences. Our analyses revealed that the gut microbiota influences host amino acid and glutathione metabolism in mice.
Tissue‐specific genome‐scale metabolic models (GEMs), transcriptomic and metabolomic analyses reveal global metabolic differences between conventionally raised and germ‐free mice and show that the gut microbiota affects host amino acid and glutathione metabolism.
A generic Mouse Metabolic Reaction GEM (MMR) is created using the mouse orthologs of human genes in HMR2.
Tissue‐specific GEMs for 28 mouse tissues are reconstructed and applied for the analysis of global gene expression data.
Microbial‐induced metabolic differences in the small intestine are simulated using the relative metabolic differences (RMetD) method.
The model predictions are validated by measuring amino acid levels in the portal vein.
The human gut harbors a vast ensemble of bacteria that have profound effects on host physiology (Huttenhower et al, 2012). Complex disorders including obesity (Ley et al, 2006; Turnbaugh et al, 2009), type 2 diabetes (T2D) (Qin et al, 2012; Karlsson et al, 2013), atherosclerosis (Wang et al, 2011b; Karlsson et al, 2012), and non‐alcoholic fatty liver disease (NAFLD) (Henao‐Mejia et al, 2012) as well as the opposite end of the spectrum, for example, malnutrition (Smith et al, 2013; Subramanian et al, 2014), have been associated with dysbiosis in the human gut microbiota. To gain mechanistic insights into the contribution of specific microbial populations to the progression of such disorders, germ‐free (GF) animals (e.g. mice and rats) have been adopted for studying the association of the gut microbiota with disease pathogenesis (Ridaura et al, 2013).
Comparisons between GF and conventionally raised (CONV‐R) mice are often used for studying the effect of gut microbiota on host physiology (Wostmann, 1981; Stappenbeck et al, 2002; Claus et al, 2008; Slack et al, 2009; El Aidy et al, 2013). Moreover, Larsson et al (2012) studied the response of the host induced by microbiota along the length of the gut in CONV‐R and GF C57Bl6/J mice and provided a detailed description for tissue‐specific host transcriptional responses.
Global metabolic differences of cells/tissues between different clinical conditions can be revealed through the use of genome‐scale metabolic models (GEMs) (Mardinoglu & Nielsen, 2012, 2015; Yizhak et al, 2013, 2014a,b; Bordbar et al, 2014; Shoaie & Nielsen, 2014; O'Brien et al, 2015; Varemo et al, 2015; Zhang et al, 2015). GEMs include the known metabolism‐related reactions and associated genes in a particular cell and tissue and serve as an excellent scaffold for the integration of omics data (e.g. proteomics, transcriptomics, and metabolomics) for increasing our understanding of the relationship between genotype and phenotype (Mardinoglu et al, 2013b). To date, simulation‐ready cell‐/tissue‐specific GEMs (Gille et al, 2010; Karlstaedt et al, 2012; Mardinoglu et al, 2013a, 2014a) and automatically reconstructed GEMs (Jerby et al, 2010; Agren et al, 2012; Wang et al, 2012; Yizhak et al, 2014a; Uhlen et al, 2015) have been used for studying the metabolism of cells/tissues in health and disease states.
In order to examine the gut microbiota‐induced transcriptional responses of the host metabolism, we performed microarray analysis of liver as well as epididymal and subcutaneous white adipose tissues (WATs) obtained from both CONV‐R and GF mice, and analyzed the global gene expression profile of these tissues together with the previously published gene expression profiles of duodenum, jejunum, ileum, and colon tissues. We created a generic mouse metabolic reaction (MMR) GEM and generated tissue‐specific mouse GEMs primarily based on proteomics data. We investigated the metabolic differences between CONV‐R and GF mice using global gene expression profiling of the host tissues and the network topology provided by the tissue GEMs, and validated our predictions by generating metabolomics data for these two sets of mice. Finally, we revealed the metabolic differences between the small intestine of CONV‐R and GF mice accounting for the content of the chow diet as well as the relative gene expression differences using relative metabolic differences (RMetD) method.
Global transcriptional profiles of CONV‐R and GF mice
CONV‐R and GF C57Bl6/J male mice were fed autoclaved chow diet ad libitum and then euthanized at 12–14 weeks of age (Larsson et al, 2012). We isolated RNA from liver as well as epididymal and subcutaneous WATs obtained from CONV‐R and GF mice and performed global transcriptome analysis (Fig 1A). Small intestine and colon have been previously removed from the same two sets of mice, and small intestine has been divided into eight whereas the colon into three equal‐sized segments (Larsson et al, 2012). Global transcriptome analysis was performed for the first (duodenum), fifth (jejunum), and eighth (ileum) segments of the small intestine and the proximal piece of the colon (Fig 1A).
We performed principal component analysis (PCA) of the transcription profiles for the tissues separately and observed a clear separation between the CONV‐R and GF mice for duodenum, jejunum, ileum, colon, and liver, whereas no separation was found for both WATs (Fig EV1). We identified significantly differentially expressed probe sets and genes in MMR, from here on referred as metabolic genes, by comparing gene expression profiles of tissues obtained from CONV‐R versus GF mice (Fig 1B, Dataset EV1). During the identification of the significantly (Q‐value < 0.05) differentially expressed probe sets and metabolic genes, we adjusted P‐values using the false discovery rate (FDR) method and calculated Q‐values. We found that ileum tissue had the largest number of differentially expressed metabolic genes between CONV‐R and GF mice, and it was followed by duodenum, jejunum, colon, and liver tissues (Fig 1B). It should also be noted that we only detected two significantly differentially expressed metabolic genes between the subcutaneous WAT whereas no differentially expressed metabolic genes between the epididymal WAT.
Comparing the differentially expressed metabolic genes between duodenum, jejunum, ileum, colon, and liver tissues of CONV‐R and GF mice (Fig 1C), we found that the expression of the nicotinamide nucleotide transhydrogenase (Nnt) gene is higher and ectonucleoside triphosphate diphosphohydrolase 4 (Entpd4) is lower in all five tissues of CONV‐R mice compared with GF mice (Fig 1D). Strikingly, we found that Nnt and Entpd4 are also the only differentially expressed genes in the subcutaneous WAT of the CONV‐R mice compared with GF mice and followed the same directional changes in the subcutaneous WAT as in all other five analyzed tissues. Entpd4 was initially named human Golgi UDPase, and it hydrolyzes nucleoside diphosphates. UDP is the best substrate for this enzyme, and its ADP activity is insignificant. Nnt is required for regular mitochondrial function, and it uses energy from the mitochondrial proton gradient to transfer reducing equivalents from NADH to NADPH. The resulting NADPH is used for driving macromolecular biosynthesis as well as for the reduction of glutathione (GSH) (Fig 1D). Here, we focused on the metabolic differences associated with Nnt due to its well‐known metabolic function.
Creation of MMR and reconstruction of mouse tissue‐specific GEMs
We constructed MMR by using the mouse orthologs of human genes in HMR2 (Mardinoglu et al, 2014a) (Fig 2A), and the resulting generic model includes 8,140 metabolism‐related reactions, 3,579 associated metabolic genes to those reactions, and 5,992 metabolites in eight different subcellular compartments. Previously, stable isotope labeling with amino acids (SILAC)‐based proteomics was generated to analyze the expression of 7,349 proteins in 28 different major C57BL/6 mouse tissues (Geiger et al, 2013) and these data cover 2,030 of the protein‐coding genes in MMR (Fig 2B, Dataset EV2). We reconstructed tissue‐specific GEMs for 28 mouse tissues by using proteomics data, MMR, and the tINIT algorithm (Agren et al, 2014) (see Materials and Methods). The tINIT algorithm allows for the reconstruction of functional GEMs based on global proteomics data as well as user‐defined metabolic tasks, which the resulting model should be able to perform. During the reconstruction of the models, we complemented 56 metabolic tasks (functions) (Agren et al, 2014), which are known to occur in all cells/tissues.
The number of reactions, metabolites, and genes incorporated in the models are presented in Dataset EV3. A total of 5,813 reactions, 4,574 metabolites, and 1,838 genes were shared across the tissue‐specific GEMs of which 2,750 (47.3%) reactions, 3,001 (65.6%) metabolites, and 669 (36.4%) genes were common to all tissue‐specific GEMs. We found that 322 reactions, 134 metabolites, and 120 genes were incorporated into only one specific GEM (Fig 2C). By pairwise comparison of GEMs, we found that each model has an average of 765 reactions (Dataset EV4), 430 metabolites (Dataset EV5), and 342 genes (Dataset EV6) different from other tissues where the muscle tissue was the one with the highest average difference (Fig 2C).
We analyzed the heterogeneity of the mouse tissue‐specific GEMs (Fig 2D) in terms of incorporated reactions, genes, and metabolites by calculating the heterogeneity degree of each model. The heterogeneity degree allowed us to capture the divergence between metabolic networks based on their constituent parameters including reactions, metabolites, and genes, and it was calculated using the average and maximum Hamming distance of the models (Ghaffari et al, 2015). Moreover, we analyzed the heterogeneity of recently reconstructed human cell‐specific GEMs (Agren et al, 2014) that have been reconstructed based on antibody‐based proteomics data in the Human Protein Atlas (www.proteinatlas.org) (Uhlen et al, 2010, 2015; Kampf et al, 2014b). On average, mouse tissue‐specific GEMs showed an average heterogeneity degree of 0.77 for reactions, 0.72 for metabolites, and 0.78 for genes, whereas human cell‐specific GEMs had an average heterogeneity degree of 0.8 for reactions, 0.7 for metabolites, and 0.84 for genes (Fig 2D). Compared with the human cell‐specific GEMs, the mouse tissue‐specific GEMs had a slightly higher metabolic uniformity and lower heterogeneity based on the incorporated genes and reactions, but they had a slightly higher heterogeneity based on the incorporated metabolites into the models.
We next incorporated the significantly differentially expressed genes between CONV‐R and GF mouse tissues and generated four functional GEMs for liver (iMouseLiver), adipose (iMouseAdipose), colon (iMouseColon), and small intestine (iMouseSmallintestine), with the latter reconstructed by merging the GEMs for duodenum, jejunum, and ileum tissues. During manual evaluation of the GEMs, previously published functional human cell‐type GEMs for hepatocytes in liver tissue (Mardinoglu et al, 2014a) and adipocytes in adipose tissue (Mardinoglu et al, 2013a, 2014b) were also used to include known biological functions to the GEMs. The number of the incorporated reactions, metabolites, and genes in the four functional annotated tissue‐specific GEMs as well as in the draft GEMs is provided in Dataset EV3. MMR as well as the all mouse tissue‐specific models are publicly available in systems biology markup language (SBML) format at the Human Metabolic Atlas portal (www.metabolicatlas.org) (Pornputtapong et al, 2015), at the BioModels database and as Computer Code EV1.
Decreased glutathione synthesis in the small intestine of CONV‐R mice
We compared the gene expression profiling in the small intestine segments (duodenum, jejunum and ileum) of CONV‐R and GF mice, and examined the changes in the expression of the genes interacting with Nnt using the network structure provided by iMouseSmallintestine (Fig 1D). We found that the expression of glutathione reductase (Gsr) which uses NADPH as an electron donor to reduce glutathione disulfide (GSSG) to GSH was also significantly higher (Q‐value < 0.05) in all three small intestine segments of CONV‐R mice compared to GF mice (Fig 3A, Dataset EV1). GSH plays a key role in reducing oxidative stress, and it can be synthesized within the cells from glutamate, cysteine, and glycine through the use of glutamate‐cysteine ligase catalytic subunit (Gclc), glutamate‐cysteine ligase modifier subunit (Gclm), and glutathione synthetase (Gss). We found that the expression of Gclc is significantly lower (Q‐value < 0.05) in jejunum and ileum, and the expressions of Gclm and Gss are significantly lower (Q‐value < 0.05) in the ileum of CONV‐R mice compared to GF mice (Fig 3A). Based on gene expression data, we observed that decreased de novo synthesis of GSH in the small intestine of CONV‐R mice was compensated by higher expression of Nnt and Gsr compared to GF mice.
The decreased synthesis of the GSH in the small intestine segments of CONV‐R mice may be due to the limited availability of the substrates including glutamate, cysteine, and glycine. Hence, we examined the expression of the enzymes involved in the synthesis and catabolism of these amino acids by differentially expressed genes in the small intestine (Dataset EV7) and using the network topology provided by iMouseSmallintestine. We integrated differentially expressed genes in duodenum, jejunum, and ileum using the lowest Q‐value for the genes and associated fold changes for studying the metabolic differences between the small intestine of CONV‐R and GF mice (Dataset EV7). Hereby, we identified significantly (Q‐value < 0.05) differentially expressed genes linked to biosynthesis of glycine (Fig 3B) and glutamate (Fig 3C) and found that there are metabolic differences in the utilization of these AAs between CONV‐R and GF mice. In contrast, we did not detect any significant change in the expression of the genes linked to cysteine except Gclc and Gclm (Dataset EV7).
In healthy animals, AAs in the small intestine are released in the plasma and utilized by other peripheral tissues (e.g. liver, adipose, and muscle tissues). Considering the down‐regulation of genes associated with de novo synthesis of GSH and of glutamate and glycine required for GSH biosynthesis in the small intestine in CONV‐R mice, we hypothesized that the plasma level of glutamate and glycine secreted from the small intestine in CONV‐R mice may also be lower compared with GF mice. Hence, we measured the level of these two AAs in the hepatic portal vein (PV), which conducts blood from the gastrointestinal tract to the liver, of CONV‐R and GF mice, and found that the PV level of glycine was significantly lower (ANOVA test, Q‐value < 0.05) and glutamate is slightly lower in CONV‐R mice compared to GF mice (Fig 3D, Dataset EV8). We also measured the level of cysteine, likewise used for biosynthesis of GSH, in the PV of CONV‐R and GF mice (Fig 3D) but found no differences, in agreement with the fact that there were no significant changes in the expression of genes encoding enzymes required for cysteine utilization (Dataset EV7).
We also searched for global metabolic differences in the small intestine by mapping the gene expression data (Dataset EV7) to the network topology provided by the small intestine GEM using the reporter subnetworks algorithm (Patil & Nielsen, 2005). Hereby, we found that there are major metabolic differences around 17 other AAs (Dataset EV9). We thus next measured the level of these 17 AAs in the PV and found that the levels of arginine, asparagine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine were significantly lower and the level of glutamine was significantly higher in CONV‐R mice compared to GF mice. We did not detect any significant changes in the level of alanine, aspartate, and lysine (Fig 4A, Dataset EV8). Our results indicate that the gut microbiota alter AA metabolism of the host.
Metabolic differences between the liver tissues of CONV‐R and GF mice
We found that the expression of Nnt was significantly higher and Entpd4 was significantly lower in the liver tissue of CONV‐R compared to GF mice (Fig 5A) and validated the expression of these genes by quantitative reverse transcription PCR (RT–PCR) methods (Fig 5B). We found that glutathione S‐transferase pi 1 (Gstp1), which has a role in GSH metabolism, metabolism of xenobiotics by cytochrome P450, and drug metabolism, is significantly higher in CONV‐R mice compared to GF mice. Notably, Claus et al (2008) measured the liver tissue level of GSSG, which is used as a substrate for the reaction catalyzed by the Gsr in CONV‐R and GF mice by employing a high‐resolution 1H NMR spectroscopic approach, and reported that the liver tissue level of GSSG was significantly higher in CONV‐R mice. Hence, we hypothesized that higher Nnt expression in CONV‐R mice might be the response of liver to the lower level of glycine required for the de novo synthesis of the GSH. Strikingly, Claus et al (2008) has also measured the glycine level in the liver tissue of CONV‐R and GF mice, and found that the level of glycine is lower in CONV‐R mice compared to GF mice. We also measured the PV level of serine, which can be taken up by the liver and converted to glycine, and found that the level of serine was also significantly lower in CONV‐R mice compared to GF mice (Fig 4A).
However, N‐acetylated AAs can also be taken up by the liver and hydrolyzed to acetate and a free AA by aminoacylases, ACY1, ACY2 (ASPA), and ACY3 (Fig 4B) which are predicted to be present in human liver tissue based on the transcriptomics and proteomics data (Kampf et al, 2014a; Uhlen et al, 2015). Accordingly, we measured the level of the acetyl‐AAs in the PV and found that the levels of N‐acetyllysine, N‐acetylalanine, N‐acetylarginine, N‐acetylcysteine, N‐acetylglutamate, N‐acetylglycine, N‐acetylisoleucine, N‐acetylleucine, N‐acetylmethionine, N‐acetylphenylalanine, N‐acetylthreonine, N‐acetyltryptophan, N‐acetyltyrosine, and N‐acetylvaline are significantly lower (Q‐value < 0.05), whereas N‐acetylserine and N‐acetylthreonine are slightly (0.05 < Q‐value < 0.1) lower in CONV‐R mice compared to GF mice (Fig 4C, Dataset EV8). We could not detect any significant changes in the level of N‐acetylasparagine, N‐acetylaspartate, N‐acetylglutamine, and N‐acetylhistidine.
Moreover, we investigated the differences in the expression of liver tissue genes between CONV‐R and GF mice. We found that the expression of Alas1 involved in glycine metabolism, Slc2a1 known as Glut1 and involved in glucose transportation, Slc16a12 involved in creatine transportation, and Hmgcr involved in cholesterol synthesis as well as Cyp7a1 and Akr1d1 involved in bile acid synthesis were significantly lower in the liver tissue of CONV‐R mice compared to GF mice (Fig 5A). On the other hand, the expressions of Sdr9c7 involved in vitamin A metabolism, Elovl3 involved in long‐chain fatty acids (FAs) elongation cycle, and Uap1l1 involved in amino sugar and nucleotide sugar metabolism were significantly upregulated in liver tissue of CONV‐R mice compared to GF mice.
Metabolic differences between the colon tissues
We found that the expression of Sardh that converts sarcosine to the glycine in the mitochondria is significantly lower (Q‐value < 0.05), whereas the expression of the genes involved in GSH metabolism including, Gsta4, Gstk1, Gstp1, and Gstt1 is significantly higher (Q‐value < 0.05) in the colon tissue of CONV‐R mice compared to GF mice (Fig 6). Even though degradation of betaine is lower, the increased expression of glutathione transferases (Gsts) may be explained by the increased expression of the Nnt in the colon tissue of CONV‐R mice similar to the liver and small intestine tissues of CONV‐R mice.
We also revealed the global metabolic differences (Dataset EV1) between the colon tissue of CONV‐R and GF mice by mapping the significantly differentially expressed genes to iMouseColon (Fig 6). We found that the expression of Arg2 involved in arginine metabolism as well as Ces1g, Aldh1a2, and Rbp4 involved in vitamin A metabolism was higher in the colon tissue of CONV‐R mice compared to GF mice. On the other hand, we found that Aldob and Aldh9a1 involved in glycolysis; Hmgcs1, Hsd17b7, Nsdhl, and Sc4 mol involved in cholesterol synthesis; Mgam and Sis involved in starch and sucrose metabolism; and Slc2a5, Slc2a9, Sord, and Khk involved in fructose metabolism as well as Ace2 transcription factor involved in the conversion of angiotensin were significantly lower in CONV‐R mice (Fig 6). Moreover, we found that the expression of genes involved in the transport of AAs is significantly lower in the colon tissue of CONV‐R compared with GF mice (Dataset EV1). Our analysis indicated that the overall central metabolism of the colon tissue may have reduced activity in CONV‐R mice compared to GF mice based on the gene expression data.
The interactions between the microbiota in the small intestine
When CONV‐R and GF C57Bl6/J mice were fed with a standard autoclaved chow diet, we found that CONV‐R mice were eating approximately 20% more than the GF mice. However, the PV level of 14 AAs and 13 N‐acetylated AAs was significantly lower in CONV‐R mice compared to GF mice (Fig 4A and C), and this may be due to the consumption of these AAs by the bacteria in the small intestine.
Bacteroidetes and Clostridium cluster XIVa are among the dominant phyla in the ileum (Zoetendal et al, 2012; Van den Abbeele et al, 2013). To understand the metabolic interactions between the gut microbiota as well as their interactions with the small intestine in CONV‐R mice, we simulated the interplay between the two key species Bacteroides thetaiotamicron and Eubacterium rectale as relevant representatives of these two main phyla in the human ileum. The Community and Systems‐level Interactive Optimization toolbox (CASINO) has recently been developed for studying the interactions between the gut microbiota and comprises an optimization algorithm integrated with diet analysis to predict phenotypes (Shoaie et al, 2015). Here, we employed manually reconstructed GEMs for B. thetaiotamicron and E. rectale (Shoaie et al, 2013) to study the interactions between the gut microbiota by using CASINO. During the simulations, we used the content of the autoclaved chow diet (Dataset EV10) and assumed that maximum 40% of the total protein may be consumed by the bacteria (set as upper bound), 5% of the total protein is transferred to the colon tissue, and the remaining proteins are consumed by the small intestine (Fig 7A) based on a previous study where the total AAs in the gastrointestinal tract of CONV‐R and GF mice were measured (Whitt & Demoss, 1975). We also assumed that 40% of the digestible and 5% of the non‐digestible carbohydrates in the diet were consumed by the bacteria in the small intestine and 5% of the digestible carbohydrates was transferred to the colon tissue (Gibson & Roberfroid, 1995).
We maximized for the growth of bacteria and predicted the amount of the short‐chain fatty acids (SCFAs) (acetate, propionate and butyrate) produced by the B. thetaiotamicron and E. rectale (Fig 7B and Dataset EV11) by setting the content of the diet as upper bound to the GEMs for bacteria (Dataset EV10). We observed that part of the acetate produced by the B. thetaiotamicron is consumed by the E. rectale and contributes to the production of butyrate in E. rectale. We found that isoleucine, proline, and valine are only consumed by the E. rectale, and glycine, serine, alanine, cystine, glutamate, histidine, leucine, lysine, methionine, phenylalanine, threonine, and tyrosine are consumed by both B. thetaiotamicron and E. rectale, whereas arginine, aspartate, and tryptophan are not consumed by neither of these bacteria (Fig 7C and Dataset EV11). It should also be noted that the gut microbiota consumed all of the available glycine, serine, and threonine during the formation of its biomass.
Metabolic differences between the small intestine of CONV‐R and GF mice
We simulated the metabolic differences between the small intestine of CONV‐R and GF mice accounting for the relative differences in the global gene expression data of the small intestine using RMetD method (see Materials and Methods). RMetD is developed to integrate the up‐ and down‐regulation of the enzymes together with the calculated corresponding P‐values into the GEMs rather than the absolute values for the expression of the enzymes.
Considering the content of the diet (Dataset EV10) as well as the calculated bounds for the intracellular reactions in CONV‐R and GF mice using RMetD (Dataset EV12), we optimized for the production of chylomicrons and compared the amount of chylomicrons and HDL produced by CONV‐R and GF mice (see Materials and Methods). We predicted that lower levels of chylomicrons and HDL are secreted by CONV‐R mice compared to GF mice (Fig 7D).
We also revealed the changes in metabolic fluxes in the small intestine of CONV‐R and GF in response to gut microbiota, and which of these differences are likely to be associated with transcriptional changes. Through the use iMouseSmallintestine, we defined a region of feasible flux distributions using uptake rates for glucose, SCFAs, AAs, and secretion rates for chylomicrons and HDL in CONV‐R and GF mice that are predicted by the RMetD. We calculated a set of flux distributions using a random sampling algorithm (Bordel et al, 2010) which allowed for identification of transcriptionally regulated reactions. Finally, we calculated the average and standard deviations for each of the fluxes carried by the reactions, and compared the changes in fluxes with the changes in the expression of the genes associated with those reactions. We found that reactions involved in fatty acid biosynthesis and oxidations are transcriptionally down‐regulated in CONV‐R compared to GF mice (Fig 7E).
The interactions between the gut microbiota, host tissues of the gastrointestinal tract, and diet are known to be highly relevant for the health of the host. In order to understand the effect of the gut microbiota on host metabolism, we revealed the metabolic differences between the continuous gastrointestinal tracts as well as liver and WATs of CONV‐R and GF mice using tissue‐specific GEMs. We observed that the ileum was the tissue that was most affected by the gut microbiota, followed by the duodenum, jejunum, colon, liver, and adipose tissues. Even though, gene expression data for each tissue were analyzed independently, we found that the expression of the Nnt was significantly higher in CONV‐R mice compared to GF mice and followed the same directional changes in six of the analyzed tissues.
We validated our GEM‐based predictions based on gene expression data by generating metabolomics data, and comparing the level of the metabolites in the PV of the CONV‐R and GF mice. Taken together, we found that the levels of the glycine and serine as well as the N‐acetylated form of these two AAs that may be taken up by the liver tissue and used in GSH synthesis were significantly lower in the PV of the CONV‐R mice compared to GF mice. Considering the lower expression of the genes involved in glucose uptake as well as the lower level of glycine in the liver, de novo synthesis of serine may also be lower in CONV‐R mice compared to GF mice. Hence, we observed that the expression of Nnt is increased in the liver of CONV‐R mice potentially due to the limited availability of glycine used as a substrate in GSH de novo synthesis.
The expression of Nnt is linked to insulin sensitivity, and higher plasma insulin concentrations have been reported in CONV‐R mice compared to GF mice (Rabot et al, 2010). Naturally occurring deletion of the Nnt in the C57BL/6J mouse strain has been associated with impaired glucose homeostasis control and reduced insulin secretion that is independent of obesity (Freeman et al, 2006). Suppression of Nnt in PC12 rat pheochromocytoma cells led to increased oxidative stress with subsequent impairment of mitochondrial function (Yin et al, 2012). Moreover, Ripoll et al (2012) overexpressed the NNT in a macrophage cell line and detected decreased levels of reactive oxygen species, indicating that NNT plays a key role in the modulation of the immune response and host defense against pathogens.
The microbiota‐induced transcriptional responses in specific fractions of intestinal epithelial cells have also been recently examined by a microarray analysis in CONV‐R and GF mice (Sommer et al, 2015). It was reported that approximately 10% of the host's transcriptome, mainly genes involved in immune responses, cell proliferation, and metabolism, was regulated by the microbiota.
The differences between the level of free AAs along the GI tract of CONV‐R and GF mice have been previously reported (Whitt & Demoss, 1975), and our analysis provided a detailed explanation of how bacteria may regulate the host AA homeostasis. We provided an explanation for the higher expression of the Nnt in CONV‐R mice, and observed that the expression of Nnt is increased in CONV‐R mice as a response to the decreased level of de novo GSH synthesis due to limited availability of glycine. We also found that microbiota in the small intestine may consume glycine as well as other AAs to support its growth and survival which leads to the decreased PV levels of the AAs and regulates the AA and GSH metabolism of the host.
Moreover, we observed that the liver and colon tissues of CONV‐R mice also responded to the lower level of glycine by higher expression of Nnt, and this indicated that the gut microbiota regulates AA metabolism not only in the small intestine but also in the liver and the colon. We investigated the global metabolic differences between the liver tissue of the CONV‐R and GF mice, and found that the expression of genes involved glucose uptake, and cholesterol and bile acid biosynthesis were significantly lower in CONV‐R mice. Previously, it has been shown that the activity and expression of the Cyp7a1, which is the rate‐limiting enzyme in bile acid synthesis, is decreased in the liver of CONV‐R mice compared to GF mice and that the gut microbiota suppress bile acid synthesis (Sayin et al, 2013). Similarly, lower expression of Hmgcr, which is involved in the cholesterol synthesis, has been reported in CONV‐R mice compared to GF mice (Sayin et al, 2013).
Our analysis also indicated that the gut microbiota regulates the host lipid metabolism. We simulated the metabolic differences between the small intestine of CONV‐R and GF mice by integrating relative small intestine gene expression data. We found that lower level of chylomicrons and HDL is produced by the small intestine of the CONV‐R. Our RMetD predictions were in agreement with experimental studies in which 40% lower chylomicron levels in CONV‐R mice compared to GF mice was detected (Velagapudi et al, 2010) and contribution of gut microbiota on the intake of fats was investigated in CONV‐R and GF mice (Duca et al, 2012).
There are several advantages of RMetD compared to previously developed methods for integrating transcriptomics data into constraint‐based models of metabolism (Machado & Herrgard, 2014). Firstly, it only considers enzyme up‐/down‐regulation rather than their absolute expression. This is based on the assumption that up‐/down‐regulation of a specific enzyme likely suggests a higher/lower flux in its corresponding reaction. This could be a better solution than inferring fluxes by comparing absolute expression level of different enzymes. In addition, RMetD pushes the flux ranges instead of the absolute fluxes. Pushing the ranges is more reasonable since in our case (and in most cases), the reference flux distribution of a GEM is unlikely to be exact, but the ranges are much more reliable. Furthermore, multiple objective products could be included in RMetD, and this could be of special interest for those using mammalian tissue GEMs since they usually have more than one obligatory functions and products.
Even though RMetD and other methods (Machado & Herrgard, 2014) for inferring flux rates from gene expression data have been developed, the correlation between the fluxes carried by the reaction and the expression of the gene catalyzing the reaction is known to be limited (Bordel et al, 2010). Changes in gene expression levels therefore only serve only as cues for the likelihood that there may be an altered metabolic flux carried by the associated reaction. To validate gene expression data‐based predictions, we performed metabolomics analysis in the PV of the both CONV‐R and GF mice and these data supported functional changes in the pathways showing altered gene expression.
The functional output and diversity of the gut microbiota are important modulators for the development of various human disorders. Alterations in gut microbiota composition and function have been shown in the pathogenesis of obesity (Ley et al, 2006; Turnbaugh et al, 2009), T2D (Qin et al, 2012; Karlsson et al, 2013) and NAFLD (Henao‐Mejia et al, 2012). Imbalances in the plasma level of glycine as well as other AAs have also been shown in obesity (Newgard et al, 2009), T2D (Wang et al, 2011a), and NAFLD (Kalhan et al, 2011). Strikingly, the plasma levels of glycine are decreased in all subjects with the above‐mentioned diseases compared to the healthy subjects. Moreover, we have recently found that the expression of the NNT is significantly increased in both subcutaneous and visceral AT after extensive weight loss in response to bariatric surgery (Mardinoglu et al, 2015). In this context, it is of interest to study the microbial AAs in the human GI tract in relation to its potential role in the development of such metabolism‐related disorders.
In conclusion, we observed that gut microbiota has a profound systemic effect on AA and GSH as well as lipid metabolism, and it is one of the major regulators of metabolism in mammals. We demonstrate that a detailed understanding of the metabolic differences between CONV‐R and GF mice obtained through GEM modeling may allow for revealing the key roles of the gut microbiota. Our findings may be used for investigating the contribution of the gut microbiome in the progression of metabolism‐related disorders as well as for elucidating the unknown etiology of such disorders.
Materials and Methods
Male C57Bl6/J mice aged 12–14 weeks were used in these experiments. CONV‐R mice were housed in individually ventilated cages, and GF mice were housed in flexible film isolators, with maximum 5 mice per cage, and fed a normal chow ad libitum. GF status was verified regularly by anaerobic culturing in addition to PCR for bacterial 16S rDNA. Light cycle was 12/12 h dark/light and lights on at 06:00. Mice were euthanized by cervical dislocation, and tissues were collected in liquid nitrogen immediately. All procedures were approved by the Gothenburg University Ethical Committee (Permit No. 339/2012).
RNA from the liver as well as epididymal and subcutaneous WATs obtained from CONV‐R and GF mice were isolated using the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany). RNA concentration and quality were evaluated by spectrophotometric analysis (ND‐1000; NanoDrop Technologies, Wilmington, DE, USA) and capillary electrophoresis on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Tissue samples with no quality issues were used in the generation of the gene expression data.
Transcriptomics data
Global gene expression profiling of liver as well as epididymal and subcutaneous WATs was measured using MoGene 1.0 ST chips (Affymetrix). CEL files for duodenum, jejunum, ileum, and colon tissue of CONV‐R and GF mice were retrieved from Gene Expression Omnibus (GEO) public repository under the accession number GSE17438.
Raw data for each tissue were preprocessed independent of the other tissues and normalized with robust multi‐array average (RMA) using Piano R package (Väremo et al, 2013). Probes which are not mapped to any gene from the analysis were removed, and the differential expression analysis for 23,736 probe sets between CONV‐R and GF mice was carried out by calculating two‐way analysis of variance (ANOVA) P‐values. P‐values were adjusted for multiple testing using the R‐function p.adjust with the method set to false discovery rate (FDR), and Q‐values were calculated. Q‐values were used for network‐dependent analysis.
During the simulation of the metabolic differences between the small intestine of CONV‐R and GF mice, gene expression data for duodenum, jejunum, and ileum were integrated. However, acyl‐CoA dehydrogenase, long chain (Acadl), cell division cycle 14A (Cdc14a), cystic fibrosis transmembrane conductance regulator (Cftr), cathepsin B (Ctsb), fructose‐1,6‐bisphosphatase 1 (Fbp1), and 6‐phosphofructo‐2‐kinase/fructose‐2,6‐biphosphatase 3 (Pfkfb3), which were significantly changed (Q‐value < 0.05) in different directions between the segments of the small intestine, were not included into our analysis.
Raw data for liver as well as epididymal and subcutaneous WATs will be deposited in GEO public repository under the accession number GSE31115. To make the gene expression data easily available to the scientific community, the data are also included into the searchable database (http://microbiota.wall.gu.se).
Liver tissue samples obtained from CONV‐R and GF mice were dissected out and immediately frozen in liquid nitrogen. Total RNA was extracted from frozen liver tissue samples using RNeasy Mini Kit (Qiagen, Hilden, Germany). About 0.5 μg of RNA was used for cDNA synthesis, using High Capacity cDNA reverse transcription kit (Applied Biosciences). Primers were obtained from Sigma, and specificity was verified by Blast and the appearance of a single product band of predicted size. qRT–PCR was performed in a CFX96 Real‐time System (Bio‐Rad). Expression levels of Nnt and Entpd4 were calculated relative to the mRNA expression of L32 and calculated with the ΔΔ‐Ct method.
Reconstruction of mouse tissue‐specific GEMs
SILAC‐based proteome reflected the proteins expressed in the specified tissues, and in total, 7,349 proteins were analyzed in 28 different major C57BL/6 mouse tissues. We reconstructed tissue‐specific GEMs for each tissue using the proteomics data (Geiger et al, 2013) and recently developed tINIT algorithm (Agren et al, 2014). During the reconstruction of the 28 draft GEMs, we used 56 common tasks that are known to occur in all human cells (Agren et al, 2014). These metabolic functions include the provision of energy and redox, utilization, and internal conversion substrates as well as the biosynthesis of certain metabolites. We compared these automated tissue‐specific GEMs and found that GEM for liver contained the largest number of tissue‐specific reactions, metabolites, and genes, which represents its unique role in overall host metabolism. Moreover, we reconstructed functional GEMs for liver and adipose tissue using the previously developed human models (Mardinoglu et al, 2013a, 2014a,b) and functional GEMs for small intestine and colon tissues. It should be noted that we reconstructed a functional generic GEM for small intestine rather than specific GEMs for duodenum, jejunum, and ileum.
AA and N‐acetylated AA level in CONV‐R and GF mice
In order to validate our model‐based predictions, we raised six CONV‐R and six GF C57Bl6/J mice and fed with autoclaved chow diet. We collected blood samples from hepatic portal vein after 12 weeks and detected the level of the AAs and acteyl‐AAs. Samples were prepared using the automated MicroLab STAR® system from Hamilton Company. A recovery standard was added prior to the first step in the extraction process for QC purposes. To remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation.
The liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) portion of the platform was based on a Waters ACQUITY ultra‐performance liquid chromatography (UPLC) and a Thermo‐Finnigan LTQ mass spectrometer operated at nominal mass resolution, which consisted of an electrospray ionization (ESI) source and linear ion‐trap (LIT) mass analyzer. The sample extract was dried and then reconstituted in acidic or basic LC‐compatible solvents, each of which contained 12 or more injection standards at fixed concentrations. One aliquot was analyzed using acidic positive ion‐optimized conditions and the other using basic negative ion‐optimized conditions in two independent injections using separate dedicated columns (Waters UPLC BEH C18‐2.1 × 100 mm, 1.7 μm). Extracts reconstituted in acidic conditions were gradient eluted using water and methanol containing 0.1% formic acid, while the basic extracts, which also used water/methanol, contained 6.5 mM ammonium bicarbonate. The MS analysis alternated between MS and data‐dependent MS/MS scans using dynamic exclusion, and the scan range was from 80 to 1,000 m/z.
Following log transformation and imputation of missing values, if any, with the minimum observed value for each compound, Welch's two‐sample t‐test was used to identify AAs and N‐acetylated AAs that differed significantly between CONV‐R and GF mice, determined independently for each dataset. A summary of the numbers of biochemicals that achieved statistical significance (P ≤ 0.05) is shown in Figs 3D and 4A and C. An estimate of the false discovery rate (Q‐value) is calculated to take into account the multiple comparisons that normally occur in metabolomics‐based studies.
Integration of gene expression data into GEM for small intestine using RMetD
In order to integrate gene expression data into GEMs, we developed relative metabolic differences (RMetD), which allow for the application of relative gene expressions between CONV‐R and GF mice rather than the absolute values, and simulated the metabolic differences using the content of the diet (Dataset EV10). First, we set the lower bounds of production of HDL and chylomicrons to 20% (arbitrary value) of their maximum production in GF small intestine model. We performed flux variability analysis for all reactions associated with the significantly (Q‐value < 0.05) differentially expressed genes in the model, and found the upper and lower bound of these reactions. A reaction could be associated with more than one gene, and these genes may have different expression trends (e.g. one gene is up‐regulated, whereas the other genes are down‐regulated). In such cases, we assumed that genes associated with these reactions are not significantly changed (Q‐value < 0.05).
Next, for reactions associated with the up‐regulated genes in CONV‐R mice, we set both the upper and lower bounds of the reactions in CONV‐R model 20% (arbitrary value) more than the bound of the reactions in GF model whereas 20% less for the reactions associated with the down‐regulated genes. By this way, reactions with up‐/down‐regulated gene expression were able to carry more/less fluxes. By adding these new constraints for each reaction in CONV‐R and GF mice together with the use of the content of the diet, we predicted the fluxes of both models by maximizing the production of chylomicrons and minimizing the sum of fluxes. For all arbitrary values, we performed sensitivity analysis by using different values and obtained similar results.
All the simulations were carried out using RAVEN toolbox (Agren et al, 2013). RMetD source code was implemented in MATLAB and RAVEN toolbox, and it is publically available at https://sourceforge.net/projects/relative-metabolic-differences/files/MiceStudy/.
Data availability
MMR generic mouse GEM, and functional GEMs for liver, adipose, colon, and small intestine as well as the other mouse tissue GEMs are publically available in the Systems Biology Mark‐up Language (SBML) format at Human Metabolic Atlas (http://www.metabolicatlas.org) and at BioModels database with accession numbers MODEL1509220000–MODEL1509220032. They are also provided as Computer Code EV1.
Gene expression data from liver as well as epididymal and subcutaneous WATs of CONV‐R and GF mice are publically available in Gene Expression Omnibus (GEO) database with the accession number GSE31115.
This project was supported by the Bill & Melinda Gates Foundation, Knut and Alice Wallenberg Foundation, Torsten Söderbergs Stiftelse, and European Commission FP7 project METACARDIS with the grant agreement HEALTH‐F4‐2012‐305312, Novo Nordisk A/S, and Novo Nordisk Foundation. We would like to thank Prof. Jan Borén, University of Gothenburg, for providing constructive comments and Doruk Demircioglu for scientific illustrations.
Author contributions
AM created MMR, generated tissue‐specific models, and performed the analysis of the gene expression data together with JN. SS performed the in silico simulations between the bacteria. MB, EL, and FB generated gene expression data for liver and adipose tissues, performed the PCR analysis, and measured the level of amino acids and N‐acetylated amino acids. PG compared the tissue‐specific models. CZ developed RMetD and performed the simulations of the small intestine. AM and JN conceived the project.
Conflict of interest
The authors declare that they have no conflict of interest.
Expanded View
Expanded View Figures PDF [msb156487-sup-0001-EVFigs.pdf]
Dataset EV1 [msb156487-sup-0002-DatasetEV1.xls]
Dataset EV2 [msb156487-sup-0003-DatasetEV2.xls]
Dataset EV3 [msb156487-sup-0004-DatasetEV3.xls]
Dataset EV4 [msb156487-sup-0005-DatasetEV4.xls]
Dataset EV5 [msb156487-sup-0006-DatasetEV5.xls]
Dataset EV6 [msb156487-sup-0007-DatasetEV6.xls]
Dataset EV7 [msb156487-sup-0008-DatasetEV7.xls]
Dataset EV8 [msb156487-sup-0009-DatasetEV8.xls]
Dataset EV9 [msb156487-sup-0010-DatasetEV9.xls]
Dataset EV10 [msb156487-sup-0011-DatasetEV10.xls]
Dataset EV11 [msb156487-sup-0012-DatasetEV11.xls]
Dataset EV12 [msb156487-sup-0013-DatasetEV12.xls]
Computer Code EV1 [msb156487-sup-0014-CodeEV1.zip]
FundingBill & Melinda Gates Foundation
This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
- © 2015 The Authors. Published under the terms of the CC BY 4.0 license


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Published Dec 21, 2017
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thank you and good luck!!!!…"
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To make great music, we need great friends. Performing magnificent repertoire in major venues requires a serious investment of time, energy, artistry, and funds. Though we tirelessly seek to maximize our income from ticket sales, engagement fees, foundations and member dues, and to minimize our expenses through judicious management and the heroic labors of an impassioned corps of volunteers, we count on the visionary support of enlightened donors.
Donations can be mailed to:
The Cecilia Chorus of New York
P.O. Box 421 FDR Station
New York, NY 10150-0421
For donation related questions, please feel free to contact us at firstname.lastname@example.com
Your inspired generosity supports the expanding scope and reach as well as the perennial vitality and relevance of what we do. You are helping create performances and so much more: artistic partnerships; community outreach; commissions from talented composers; regional premieres of neglected masterpieces; life-changing performance opportunities for budding choral singers; exhilarating musical encounters for under-served or inexperienced listeners; exceptional showcases for emerging soloists on the cusp of important careers; training and mentoring of promising conductors.


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Graduate Degrees & Programs
Embodied in a graduate program are the opportunity and responsibility to conduct research leading to new knowledge or the application of existing knowledge in new and novel ways. The following is a representative list of areas in which faculty are actively researching:
- Information Assurance and Computer Security
- Large-Scale Data Management
- Games and Virtual Environments
- Fault Tolerant Systems
- Artificial Intelligence
- Computational Intelligence (Artificial Neural Networks, Neuro-Fuzzy Systems)
- Evolutionary Computing
- Bioinformatics
Enrollments in the department's graduate programs are large enough to make possible the vital interchange of ideas among students and between students and faculty that is necessary in graduate programs, and sufficiently small to permit close faculty-student relationships. Interdepartmental cooperation is an important factor on the Idaho campus.


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Create an Account - Increase your productivity, customize your experience, and engage in information you care about.
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The purpose of the committee is to:
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The Monroe Lodging Tax Advisory Committee Members are currently:
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The 2020 Lodging Tax Application submittal period will open soon.
For more information, please contact Becky Hasart at (360) 863-4518


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If you want to be liked by your customers, do it right, from the start.
That applies to any business endeavor, doesn't it? It certainly describes the approach we take at ActiveData. Find out how we can help you right from the start to increase the rewards and reduce the hassles of a world gone exuberantly and irretrievably mobile…
It's a world changing so fast that at times it can seem downright intimidating—especially to someone already spread thin trying to manage and grow their business or organization. Sound familiar?
For example: beyond optimizing your communications for a variety of mobile platforms, what realistic goals should establish? What mobile marketing strategies should you employ? What form should these communications take? How can you be certain you're using the newest technologies and strategies? How can these programs support your brand and social media efforts? And how can you learn from the data feedback you receive to continually improve the ROI on your mobile marketing investment?
More than answers. Proven solutions.
We can answer your questions—and lower your blood pressure—with the same kind of customized, revenue-producing solutions that are generating new customers and building customer loyalty right now for businesses and organizations across the nation in more than a dozen industries.
Check out what we do and what makes us unique. Then contact us today, We'll show you how we can get your mobile marketing programs up and running faster, easier and more affordably than you might think possible. Remember, do it right from the start, and the possibilities are infinite.
Every member of the ActiveData team is dedicated to helping our clients succeed through the power of mobile marketing done right, from the start. What makes us unique is how we deliver on that promise, project after project…
1. No off-the-shelf solutions. You'll find ActiveData's fingerprints all over everything we do for and request of our clients. All of it designed first and foremost to protect the consumer and to protect your brand.
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3. You'll lack for nothing. Our approach and abilities encompass brand support, strategy, mobile -friendly (optimized) design, data analysis, personal follow-through and continual learning—all of which leads you to happier, more loyal customers and new revenue for your bottom line.
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If this is the kind of all-encompassing, dedicated, team approach you're looking for, it's easy to get started…right here.
Infinite possibilities, all over the map…
We've been at this mobile marketing thing long enough now that we've developed an expertise in certain core industries. And yet, virtually every day we start a new mobile marketing relationship with a client in a different field of endeavor. Not only is that exciting for us, but it also means you can be confident we'll know how to make mobile marketing work for you, too. Today, we're honored to serve great clients across the nation in two dozen distinct categories—and counting:
If you're ready to make the power of mobile marketing a reality for your business or organization, we're ready to help with the real world experience and priority marketing solutions you'll need and want.The possibilities to build revenue, enhance loyalty, expand your social reach, strengthen engagement and support your brand are infinite and, literally, all over the map. But only if you know how to do mobile marketing right, from the start.Where does that start? Right here!


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Adamawa United FC is based in Yola in northeastern Nigeria. According to http://www.nigerianationalleague.com/, Adamawa United FC ended the season as number 8 of 9 teams in "Division B" of the Nigeria National League 2012. In other words, the club was relegated and will play at third level of Nigerian football next season. The club scored 19 goals during their 16 matches. I've been able to find the scorer of 15 of these goals.
It is very difficult to find information about this team. I know that Tijani Dansabe, Sadiq Lawal and Abubakar Shehu scored at least two goals each. Yakubu Auta (winger), Hamidu Gambo, Isah Garba, Sadiq Lawal, Tunde Oladapo, Azeez Abiola Safaru, Abubakar Shehu, Lukman Surajo and Ismaila Yahaya are other players I have noticed.
Ajiroba-Ooni FC is based in Ile-Ife in south-western Nigeria. According to http://www.nigerianationalleague.com/, Ajiroba-Ooni FC ended the season at the rock bottom of "Division D" of the Nigeria National League 2012. In other words, the club was relegated and will play at third level of Nigerian football next season. The club scored eight goals during their 16 matches. According to my own statistics, Ajiorba-Ooni FC scored nine goals and I have been able to find the scorer of all these goals.
Yusuf Oyediran is arguably the most interesting player at the club. The young goalkeeper participated for the Nigeria U-20 national football team during the Cape Town U-20 International friendly tournament.
Taiwo Amusan scored two goals during the season. According to Completesportsnigeria, Amusan is a talented 18 year-old youngster who can play in several attacking positions. Amusan is apparently the brother of petit striker Adewale Sunday Amusan, formerly of FC Koper (Slovenia) and currently playing for Wikki Tourists FC.
Funsho Alamayo started the season as the main striker of the team but joined top level side ABS FC in May 2012.
Other players I have noticed are Adekunle Busari, Sunday Ndubuisi, Austin Iluebbey, Saheed Kayode Lasisi, Arowolo Olamide, Shehu Shobayo, Nurudeen Ajibola and Salaudeen Sileola. Shobayo scored two goals during the season.


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True luxury icons. Magnificent and rare stones are cut and set into innovative and voluminous designs for a statement of prestige.
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Ricky Martin, Gloria Estefan and Arturo Sandoval are a few of the artists who will perform in Washington next week during the White House's celebration of Latin music.
"Música Latina: In Performance at the White House," will be hosted by President Barack Obama and the First Lady Michelle Obama.
The event will take place in the East Room of the White House on Monday, Sept. 16 and will air next month on PBS.
Natalie Cole, Lila Downs, Raul Malo, Prince Royce, Romeo Santos, Alejandro Sanz and Marco Antonio Solís will also perform.


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[ Index ] [ The 7.5 Nano Reef ] [ The 15 Nano Reef ]
Recent Entries
May 19, 2003
Not much happening...
The tanks are both doing well as usual. The new orange/red ricordea in the 15 are absolutely huge, and the purple montipora are happy as can be. They are still floating in the basket though because I haven't had any time to mount them. The zoanthids in the 15 have been growing very quickly too, and I was thinking of moving some to the 7.5.
Anyways, that's about it. I'll try to get some things done in them this week, and when I do I'll post some more updates and photos in the journals.
Posted by Christopher at 09:04 AM | Comments (13)
May 11, 2003
Both tank's updated
Both tank journals have been updated today - with photos!!!
Go take a look and post your comments for me :-)
Posted by Christopher at 01:23 PM | Comments (3)
May 08, 2003
New Corals!
I received a package from The Logical Reef yesterday morning. I purchased some new corals for the 15 gallon, and perhaps one for the 7.5. Right now they're all in the 15 though. I'll get some photos of them today and post an update in the 15 gallon nano reef log, now that they're open.
Check back soon!
Posted by Christopher at 10:12 AM | Comments (39)
May 05, 2003
15 Nano Reef update
I posted some info and cool photos about the GSP plague in the 15 gallon nano reef. Take a look!
Posted by Christopher at 07:50 PM | Comments (40)
May 03, 2003
Today's update
The 7.5 Nano Reef journal has been updated with a new addition to the tank and observations.
I'm preparing this week for a local frag swap on the 10th, where I'm hoping to pick up a new coral or two, and sell off the liverock from my now taken apart 90 gallon system.
I sure miss the 90 gallon reef. I had to take it down though...no apartment landlord would allow a 1.5 ton aquarium, and there's no way I'll be able to move it when I leave home. I'm content with my nano reefs though! Here's a flash back to the good old days of the 90 gallon:
Posted by Christopher at 10:35 PM | Comments (13)
May 02, 2003
Desktop Wallpapers
I uploaded a few desktop wallpapers from some of my photos in the 15 gallon. If you're interested, I have them in 800x600 and 1024x768. If you want a larger size, post a comment to this or email me, and I'll try to get some made. Post your feedback on them if you like.
I'll get some small thumbnails of them and a wallpapers page created sometime soon.
Posted by Christopher at 12:25 PM | Comments (17)


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Allcare Home Services & Pest Control
Pest Control Packages for Three to Four Bedroom Homes! Only $89 to Rid Your Home of Ants, Fleas, Cockroaches and Spiders (Valued at $320). Upgrade to Add Mice and Rodents to the Mix for only $109 (Valued at $470). Includes Homes within 50km of CBD
- Highlights
- Fine Print
- Company
Help get rid of nasty bugs and spiders with one of two pest control packages from the skilled team at Allcare Home Services and Pest Control
Option One: Only $89 to target ants, fleas, cockroaches and spiders. Valued at $320
Option Two: Only $109 to add mice and rodents too with baits placed in the roof and sub-floor areas. Valued at $470
Allcare will services all three to four bedroom homes within 50km of the Melbourne CBD
Includes pest control treatment of the house perimeter, exterior and interior
Each technician is professionally trained, insured, background checked and certified so you know you're in safe hands
What We Like
Allcare Home Services and Pest Control is one of Australia's leading cleaning companies offering the highest quality of service for residential pest control and cleaning services throughout Melbourne
Your package is valid for both inside and outside of your home!
Offer expires on 30 April 2014 (bookings must be made by 30 March 2014)
Bookings are essential and subject to availability. Not valid on public holidays
Members can purchase and redeem one voucher for themselves and up to three as gifts for different recipients
Maximum of one voucher per household
Valid for all standard sized three to four bedroom houses and apartments (residential only)
Fences will occur an additional charge of $50
Valid only for the perimeter of the house inside and outside
Valid for up to 50km radius of the Melbourne CBD. A surcharge of $50 will apply for anything further. If unsure, please contact Allcare Home Services and Pest Control directly before purchasing the deal
Residents and pets must not be in the house for four hours after the treatment has been applied
Allcare Home Services and Pest Control must be able to access under sub-flooring
Severe infestations will incur a surcharge, please enquire with merchant at time of booking
Parking must be available for the pest controller either on your premises or available just outside your building
24 hour cancellation policy applies, non-attendance and late cancellation voids voucher. If the pest controller is unable to gain entry to your premises at the time of booking, your voucher will be forfeited
Not valid in conjunction with any other offer and not redeemable for cash
Redemption instructions can be found on your voucher


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We focus on providing legendary customer service, outstanding selection, and honest, helpful advice. With seven locations spanning Western Montana, Montana Ace has the buying power to keep prices low and selection high. We hire talented people with real life experience, and we strive to exceed your expectations every day.
Doing the right thing, it's the Montana Ace way.
Join the conversation, and follow us on Facebook, Twitter, Instagram and YouTube. You'll get insider tips and advance notice of fun things like workshops, seminars and sales. Plus, being our friend has it's advantages. You'll receive exclusive Friends and Followers special offers and coupons.
See you online!
Montana Ace participates in the national Ace Rewards program. Earn points with every purchase you make for cash rewards to spend in the store. Plus, your Ace Rewards card gets you access to Instant Savings, advance notice of sales, and special offers delivered directly to your inbox.
Not a member yet? Or would you like to manage your Ace Rewards account?
click HERE
Time flies! Montana Ace will honor Ace Rewards coupons for 3 months past the stated expiration date. See stores for complete details.
Are you 65 or older? Every Wednesday is senior discount day. Seniors receive 10% off all regular priced items on Wednesdays.
FREE and EASY to join, the Montana Ace Veteran's Club entitles eligible Veterans and Active Duty Military personnel to 10% off regular priced items EVERY DAY. Sign up is easy: pop in to any Montana Ace location, show us a valid Military ID (DD214 w/ID, VIC, or driver's license with Veteran designation) sign the form pledging to play by our rules, and VOILA! Your discount is active and ready to use. See stores for complete details.
If you need to return an item to Montana Ace, we will fix it, exchange it, or refund your original purchase price, your receipt guarantees it!
The following items do not qualify for additional discounts during our storewide sale events:
The following items qualify for a maximum discount of 10% off regular price during our storewide sale events:
Manufacturer coupons are gladly honored at Montana Ace. Unfortunately, we can not honor expired Manufacturer coupons.
Our stores are open 363 days every year. We are closed on Thanksgiving and Christmas.
Memorial Day through Labor Day our stores are open 7am-10pm
Labor Day through Memorial Day our stores are open 7am-9pm
Power Pros hours vary by location. Please contact store for details.


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What is the deal these days? It is as if any sort of backbone that America had was thrown away. People these days in America, and the world for that matter, simply have no backbone or, to put it more bluntly, no cajonies. They are offended at everything, never want to take a stand and simply let people walk all over them.
These days a big personification of a Wuss is the President himself. George W. Bush. The guy can't seem to do anything without apologizing for it. The Liberals will out and out lie about him and he does nothing. In fact, he usually just apologizes for nothing. Take the current flap about Federal Attorneys. So he fired them. SO WHAT? Clinton fired ALL of them. A President can do that. What did Bush do? He apologized for how it was done. Illegal immigration? Bush talks about amnesty. Sandy Berger steals top secret documents from the Federal archives and Bush does nothing and says nothing. Get some balls Mr. President.
Liberals are not much better. They only want appeasement for anything and everything. Clinton pretty much did nothing whenever we were attacked in the 90's. Bin Laden even said that because of what the U.S. did not do in the 90's was why he saw attacking us as the perfect opportunity. He knew that these days the U.S. is just a bunch of wimps. The first sign of trouble in a conflict and Liberals want to surrender. They are worse than France!
Jimmy Carter has turned into probably the biggest Wus of all the Presidents. No dictator can do anything wrong. He goes and has tea with Fidel Castro and Kim Jong-Il, among others. We all saw how well Carter handled the Iranian Hostage crises.
The media plays a big part in this as well. 15 British sailors are kidnapped by Iraq and do you see the media saying anything negative about them? They are praising Ahmadinejad and the Iranian people. Not a thing was said of Iran's violation of the Geneva Convention. The media also never seem to condemn anyone. Shortly after 9/11, Reuters news service announced they would no longer use the term "Terrorist" when talking about, well, Terrorists. What a joke.
Speaking of those 15 sailors….what did Britain do? NOTHING. Tony Blair = WUSS. The Islamic Terrorists around the world are going to get more and more emboldened and know they can do pretty much anything and the rest of the world will do nothing.
Now, with all that being said, I am not advocating war or aggression for no reason but come on…can't we at least say SOMETHING and not just get walked all over by everyone? Can we at least speak softly but carry a big stick instead of apologizing for talking too loud and then appease appease appease?
I didn't mean for this post to be so political, I wanted to write more about things in every day life as well. I mean, they seem to call counselors into schools and work places at the drop of a hat. Suck it up!!! These days people go see Shrinks as much as they change their underwear. I'm not saying they aren't needed…at times…but it has been taken to excess. These days if you DON'T have some "problem", "issue" or "wound" and go to a shrink and get drugged up then you are not normal. If you do mess up all you have to do is apologize, cry, go to rehab and then admit that you were bullied on the playground when you were little and you get a pass. HEY! Suck it up, take some responsibility and move on. The world doesn't revolve around you!!
TV is not much better. People don't like it when Simon is so mean to people on American Idol. Hey, if they suck, they suck. Deal with it. The very fact that so many people get caught up and sucked into watching this show says a lot about our society. It says, in big lavender letters, GAY! (Don't anyone say anything about me watching America's Next Top Model either because there are hot chicks on that show!) 😉
There is a phrase I use with my kids all the time. "Dry it up". They fall down and start crying I first make sure they are not hurt then I tell them to dry it up. I don't rush over and make a big fuss over them and coddle them. I am hard on my kids. I push them to go beyond what they think they are capable of. I want them to look back when they are older and know that I pushed them to exceed. I tell the story often that when my daughter was 2 or 3 she didn't want to go down a slide. I made her because I knew she would have fun. She was scared but then loved it. I did the same thing when teaching her to read. When she wanted to stop I kept going. I showed her what was beyond her limits. I expect a lot out of her and don't apologize for it. As I said before in my overprotective parents blog it's the feminization of our society approach that I don't want to see come near my kids. Go mess someone else's kids up.
These same wimps want to get rid of things like Dodge Ball or even Tag at school. My friend at work said he can't even cheer for his kid as his little league game because they don't want to create a "negative atmosphere". In fact, some cities are banning "chatter"at baseball games so you can't yell the old "Heeeeeeyyyy Batter Batter Batter SWING Batter!". GIVE ME A BREAK!!! That has Liberal weenie written all over it. The same wimps who say things like "Everyone is a winner so let's not keep score" or "there are no losers so everyone gets a trophy!". These are probably the same people who want to stop having a Valedictorian or a Salutatorian at graduations.
So, let's all dry it up, get some balls and deal with these things as they should be. If we don't, if we keep going down this road, in 20 years we will have no country to call home because we will have given it over to Islamic fascism and wimps because we simply don't want to offend anyone.
Now, over in Britian they are proposing that kids don't raise hands to answer questions in class. Education Secretary said "We need to make sure that no one is left behind at any point…" Yeah, that's right. Don't praise kids who study and are actually smart! What a joke. Remember, it's all about "fairness".


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VIDEO: An all-access look at the 2015 Hall of Fame ceremony
Van Gundy unsure of Jennings' role | Report: Nets, YES Network nearing new deal | Report: Pelicans add Douglas-Roberts
No. 1: Van Gundy unsure what Jennings' role will be with Pistons — By most accounts, Detroit Pistons point guard Brandon Jennings is recovering nicely from the Achilles injury he suffered last season than ended his 2014-15 campaign early. He should suit up at some point this season. While that's good for Jennings, what may be bad for him is the Pistons' new point guard depth chart given that the team re-signed Reggie Jackson to a large deal this summer. Kevin Bull of the Detroit Free Press details how coach Stan Van Gundy is unsure what Jennings' role will be in Detroit come 2015-16:
Detroit Pistons point guard Brandon Jennings is not expected back from his Achilles injury until mid- to late-December — at least a month and a half after Detroit's season starts Oct. 27.
Pistons coach Stan Van Gundy said Thursday what role Jennings will play is unclear.
"That's really hard to determine right now," Van Gundy told WMGC-FM (105.1). "We don't know where Brandon's gonna be health-wise. Right now, he's still recovering."
"If he comes back and he's the Brandon Jennings that we had last year, I think those guys can play together quite well," Van Gundy said. "Reggie is big enough (6-feet-3) to guard guys off the ball and everything else. It just gives you two playmakers on the floor together. I think they can be pretty dynamic, but we'll just have to see where Brandon is when he comes back."
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Texas A&M football is a six-point favorite over Arkansas as both the Aggies and the Razorbacks look to maintain their hot starts
The Texas A&M Aggies are 4–0 straight up and 2–2 against the spread in their last four games against the Arkansas Razorbacks. The Aggies will look for another win over their conference rival Saturday night at AT&T Stadium in Arlington, Texas.
Texas A&M is a six-point betting favorite at sportsbooks monitored by OddsShark.com. The opening line had the Aggies going off at just minus-three, but action on Texas A&M has moved the line three full points.
Hot starts have been commonplace for the Aggies in recent seasons as they has gone 12–0 SU and 9–3 ATS in their last 12 games played in August or September. This season has been no exception as Texas A&M has jumped out to a 3–0 SU and ATS start with impressive wins at home against UCLA and on the road against Auburn. With games against Tennessee and Alabama looming in October, the Aggies hope to avoid a costly slip-up this week.
Texas A&M was a 2.5-point underdog in last week's 29–16 win over Auburn. In the Aggies' last eight games coming off an upset win, they are 7–1 SU and 6–2 ATS per the OddsShark College Football Database.
Arkansas stumbled out of the gate with an uninspiring 21–20 home win over Louisiana Tech. But the Razorbacks have looked sharp since with a 41–38 upset victory on the road against TCU as a 10-point underdog and a 42–3 rout at home against Texas State last week.
Since starting the 2015 season off 2–4 SU and 3–3 ATS, the Razorbacks are 9–1 SU and 7–3 ATS over their last 10 games. They are 6–1 ATS in their last seven games against teams with winning records.
The total for Saturday's betting matchup is 49.5 points. The UNDER is 7–1 in Texas A&M's last eight games and 9–3 in Arkansas's last 12 conference games.
Four of the last six games between these two teams have been decided by a touchdown or less. Texas A&M has won each of the last four games against the Razorbacks by at least seven points, but on paper this one doesn't figure to be a blowout in either direction.


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Thurgood Marshall, the first African-American Supreme Court Justice, battles through one of his career-defining cases.
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With 2018 well under way, and the second legislative session of the new government about to begin, it's a good time to take stock of the changes that have been made, and how we will focus our efforts over the year ahead. As a history professor, I also cannot pass up on the opportunity to place this moment into historic context as we cannot know where we are going until we remind ourselves of where we have been.
Thanks to the BC Federation of Students (BCFS) for inviting Federation of Post-Secondary Educators of BC President George Davison to address attendees at their annual general meeting Saturday, January 13. Here's George's speech to BCFS members, underscoring the importance of reducing student debt and addressing post-secondary system underfunding.
This has been quite a year for our Open the Doors campaign. From our Days of Action in January to a new government sworn in after 16 years, to Minister Melanie Mark meeting with the FPSE Presidents' Council in Victoria, 2017 has been a whirlwind of activity and change.
Subscribe for updates about BC post-secondary education from Open the Doors.


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On June 15, I'll be teaching a class at VIA on making and using faux coral and ivory. Polymer clay is such a great mimic that a coral vendor actually asked me where I bought the materials for this 100% polymer necklace. I was thrilled! Polymer coral involves using surface textures and paints applied to the clay body to imitate the pits and variations of the real deal.
These two pieces are made of faux ivory adorned with simple stripes and canes.
In this class, we'll make the ivory and coral clay bodies and use texture tools to create design elements to make beads; we'll also create a couple of simple canes to use.
We'll use moulds to make an ivory pendant, which could be used by itself or combined with other beads.
To sign up for Ethical Amulets, go to http://viaartisticapdx.com/