text:
Are you looking for the best Medigap insurance in Glassport, PA? We can help you find affordable and quality Medicare Supplemental Insurance in Pennsylvania. You can either call 888-602-5343 or enter your Zip Code above and fill out the form. We will help you find the best Glassport, PA Medigap insurance options.Buying Medigap, or Medicare Supplemental Insurance, is the key for many people over age 65 to be able to afford to use their Medicare coverage. Medigap is a separate policy that is sold by private insurance carriers that adhere to Pennsylvania laws.
For the most part, Medigap covers co-insurance, co-payments, and even includes medical coverage if faced with an emergency when traveling outside of the United States. Older Medigap plans also covered deductibles, though those were mostly phased out in 2010. Medigap covers costs seen in Medicare Part A and Part B Plans. Medicare Advantage plans are also available though not in conjunction with Medigap benefits. Likewise, be aware that it is illegal to sell a Medigap holder Medicare Savings plans.
The reason people in Glassport, PA buy Medigap is to cover where an individual's money will fall short. Buying a Medigap policy will require monthly premiums in addition to Part A and Part B Plan premiums. Though, the fact is that there are still ways to save on Medigap coverage.
For one, apply for policies while in the first 6 months of your Medicare Part and Part B plan policies. During this period, guarantee issuance is extended to anyone over the age of 65 within the first six months. It means that there are no denials or increases in premiums because of pre-existing conditions, based upon past medical history, or current medical problems that spring up.
If an individual waits they may pay higher rates and fees for extending past the deadline. It is better to shop around and compare quotes and do so during the open enrollment period. The majority of Medicare Supplemental insurance coverages are standard and demarcated by letters A through N. Each plan covers benefits but have added benefits. For instance, part D covers prescriptions.
That means that while cheap Medicare Supplemental Insurance plans A through G charge higher premiums, costs that are out-of-pocket are not as expensive as for Plan K through Plan N. Those carry lower premiums and like benefits but at the trade-off of costing more at the time of service.
Employer Retiree Benefits
The next way to save money beyond following the open enrollment dates is to find out if a former or present employer has a Medigap policy of its own. In general, these will be less costly than an individual policy that you buy on your own. Though, the warning and word to the wise is to carefully consider if the company is financially stable or not.
Determining Eligibility
Whenever purchasing many types of Medicare plans, you actually cancel the old policy before instating the new policy. That is not the case where Medicare A and B are concerned. The reason, recall, is that inexpensive Medicare Supplemental insurance plan is meant to fill the gaps of conventional original Medicare policies.
It requires still being covered under Medicare. The other benefit is to know when shopping around that the policies are the same whether you buy with Humana or Aetna or BlueCross BlueShield, for instance.
What is worth looking at is the reputation of the health insurance company, the quality of the coverage they offer, and the credit worthiness rating of the insurance company. The reason to look at the credit worthiness of the company offering up the insurance is to ensure that they are likely to have the money to pay out benefits and claims as agreed in the policy paperwork.
In addition to looking for insurance carriers that have an A or greater rating in the creditworthiness area, it is important to find out about the customer service and the customer satisfaction ratings of the companies you are comparing as well. It pays to have the company that pays claims within a timely manner, without a heroic effort on your part or the part of your doctors.
Otherwise, you will spend time having to contact the Pennsylvania insurance department or commissioner to file official complaints to get the claims paid within the acceptable two-month window. All of the Plan C Medigap coverage will be the same as that same plan from another insurer. Look at service quality and price. Look at comparing insurance company offerings to locate a cheap medicare supplement insurance plan in Glassport, PA .
You will also need to look for separate prescription drug coverage, which is designated by Medicare Part D. This will add in an additional premium in addition to other costs. The key is that the majority of people who qualify for cheap medicare supplemental insurance are 65 and older, and look for their Medigap during open enrollment.
In addition , the other requirements stipulate Medicare Part B and Part A participants are eligible. Once you choose a inexpensive Medigap insurance plan it is as if it is written in stone and can not be altered.
To be eligible for cheap Medicare Supplemental insurance plans in Glassport, PA apply for coverage offered by insurers within your state. All policies are available in each state, though most states will have one single policy available for Medicaid plan holders.
Everyone at age 65 and older and who is also enrolled in the Medicare Parts A and B is eligible to purchase a supplemental plan. It is advisable to decide on a plan during the 6 months of open enrollment to pay the most favorable policy premiums. When comparing costs during the open enrollment, it means pre-existing conditions are not factored into the pricing for the premiums. It is the hands-down best way to save money on a Medigap Supplemental plan in Glassport, PA .
Always find out the Pennsylvania laws prior to shopping. They can vary a bit, though it is important to recall that the policies are uniform across states. Use online free quotes searches to find all of the options available to you. Narrow it down based upon credit worthiness of each of the insurers, along with their quality of care and their speed of paying out claims.
More Pennsylvania Cities We Cover:
More Tips About Glassport, PA Medicare Supplemental Insurance
Picking the proper wellness insurance policy is 1 of the biggest decisions you will make in your daily life. In get to know what to seem for in a wellness insurance coverage firm you must teach your self and find out about the health protection presented by different insurance coverage businesses. Utilize the guidance from this write-up and you will have no trouble selecting the right insurance plan for you.
When searching for affordable insurance policy make sure you get prices from all available options. Every single company has diverse costs depending on your history. The greatest wager to make sure that you help save the most funds is to assess rates. With that being said, make certain you are relaxed with the selection you make and the kind of coverage you receive.
When considering a overall health treatment insurance coverage program from your employer, be weary of certain incentive primarily based programs that try to entice you. These could seem appealing at very first, but there might be hidden costs or decline of previous benefits. Constantly go through the deal thoroughly and do not be concerned to request concerns about everything you are unsure about.
Dental insurance policies is some thing that can prove to be quite useful. Purchasing dental insurance policy imply that when you want a cleaning or anything at all else done at the dentist, a proportion will be paid out for. If you have youngsters, dental insurance is a have to, because the cost of likely to the dentist can get really large.
Check out your health insurance policy plan to make positive your partner receives the same coverage you do. Some insurance policies businesses will only supply the identical coverage for your husband or wife if you pay out a considerably greater quality. Look for for a coverage that provides you the two the identical advantages, for a price that you can pay for.
Use the assets available to you. There are many websites online that allow you to privately enter your family's information, and use it to assess overall health insurance policies policies and charges. This can be extremely advantageous to people who could not have the time to store around for this data on their personal.
1 of the most straightforward and but valuable ideas you can get about obtaining a wellness insurance coverage coverage for you and your family members is creating positive that you get one particular that you can manage to pay out the premiums for. If you are on a limited price range, it is much better to get a coverage in which you can pay out the premiums for than getting rid of the plan owing to non-payment.
Read through your well being insurance coverage very carefully before you go out and get eyeglasses or get your tooth set. Most healthcare insurers offer dental as a separate policy, and several do not offer you eyesight insurance coverage at all. Far better to know up entrance whether your eyesight care is protected than to be greeted with a bill from the eye medical doctor and not be capable to pay it.
If your wellness insurance coverage will come by way of your employer, you evidently never have much choice about who insures you and your loved ones. You do, however, have some alternatives about what options you want. Be as active a customer of your healthcare insurance policies, as achievable. Get the time to recognize the philosophical and genuine variances among HMOs and PPOs and the attendant variances in value composition. You need to have to be armed with this information, in buy to make smart choices about your health care insurance policy.
You in no way know when sickness or injuries will strike, and the charges of treatment method can be financially devastating. It is sensible to carry overall health insurance policies to lessen the likelihood that sudden medical expenses will lead to financial hardship. From bare-bones guidelines to all-inclusive options, there is a wellness insurance coverage coverage to match any scenario.


text:
Esther Jenner, Caitlyn Jenner's glamorous mother!
Why didn't anybody ever tell me about the Waffle House waitress circa 1968 glamour of Esther Jenner?! My eyes may have graced against the beauty of Esther Jenner before since she's done the talk show rounds many times (She was even in Caitlyn's big Diane Sawyer interview. How did I miss that?), but I never sat up and took notice until a couple of days ago when Access Hollywood talked to her about the unveiling of Caitlyn Jenner. Esther has also been on a few episodes of the Keeping up with the Kardashians before and even though I fill my head with a whole lot of steaming hot TV trash, I draw the line at that mess. I have SOME standards (not really). And yeah, watching a few episodes of Pimp Mama Kris' circus of fame whores while in a vulnerable state (read: drunk, stoned and lonely) doesn't count!
Memaw Esther is the chain-smoking, Pimp Mama Kris-hating mother of Caitlyn Jenner who probably still says, "I'm just here to get my hair set, honey," to her hairstylist, keeps her favorite jewelry in an old round yellow Jean Nate powder box and paints her brows on with a Mary Kay pencil. After Caitlyn Jenner's debut, Memaw Esther talked to Access Hollywood. Memaw Jenner is here for Caitlyn Jenner, but says that she's not ready to stop calling her "Bruce" just yet. 88-year-old Memaw Esther has been calling Caitlyn "Bruce" for a million years, so it's understandable and I'm sure she'll start calling her Caitlyn eventually. I mean, she even calls her that while talking to Access Hollywood.
"She's beautiful! I was there last week, well actually, I got home a week and a half ago. And I met Caitlyn. She is… so much more at ease. I still have to call him Bruce. His father and I named him that. It's going to be a struggle for me to get used to the change, but happily so… I'm very happy for him – or her!"
And like I said before, she thinks PMK is a monster. What's not to love?
Pic: Wenn.com/ABC

subset: commoncrawl


text:
Ello ello, what the fuck is going on here? We reckon the director 'Price James' hasn't a clue either. Just when you thought the Selfridges' 'skatebored' fiasco could get any worse, Dazed and Definitely Confused just released this top effort making skateboarding a laughing stock once again.
"Let the board games begin" say Selfridges. Indeed. Anyone involved in this who come from London's skateboard scene should be highly embarrassed.
More fashion twat related belly-laughs here if you missed them.


text:
April 1, 2015
Oh man! Another gap between episodes? Yes. Yes it's true. Both Jason and I have been traveling a bit this Spring so our schedules have been a little wonky. I just got back from Emerald City Comic Con in Seattle and Jason ran off to the Twin Cites for a bit, so we were literally running in opposite directions! But don't worry, we will be reunited this Easter weekend as he is a guest on a panel that I'm moderating this Friday at WonderCon in Anaheim! (more on that below!)
Whenever there is a gap between episode it just means that there's even MORE to discuss each time we sit down together.
In this episode we discuss:
All that and more in episode 120!
If you find yourself at Wondercon 2014 in Anaheim this weekend be sure and check out our panel:
Toy Collecting in the Modern Age: The High Price of Having Fun
Friday April 3, 2015 1:30pm – 2:30pm
Are mass-market collector toys dying? The price of raw materials and costs of the production process continue to skyrocket. Action figures that were $7.99 five years ago are now in stores for over $20. At that price can you still be a "completist?" Is Kickstarter the better way to go for an up-and-coming toy company to get figures in the hands of collectors? Are toy collectors prepared for the onslaught of all of the new Star Wars figures that will come from the new movies? A panel of toy industry insiders and collectors examine just what is going on today and where it's all going in the future. Moderator Daniel Pickett (ActionFigureInsider.com, host of Geek Shall Inherit podcast) is joined by industry experts Justin Donaldson (collector, director, The Tournament of Nerds), Jim Fletcher and Kevin Kiniry (DC Collectibles) Jason Lenzi (co-founder, Bif Bang Pow!), Scott Neitlich (director of play, Jakks Pacific, collector), Jeff Trojan (Playmates Toys) and David Vonner (senior designer/product manager Spin Master).
Podcast: Play in new window | Download (Duration: 1:44:46 — 119.9MB)
Posted by Daniel Pickett • 1 Comment
1 Comment »
BLARGH! I need my GSI Fix! Miss you guys…


text:
American Church Adapts to Self-Centered Culture
The expression "secular church" may be a contradiction in terms, but it is what many sociologists say is emerging on the American religious scene.
The secularization process that has transformed Western civilization over the past two centuries has taken different paths in Europe and in America. In Europe, secularization has meant large-scale defection from religion and the appearance of many empty church buildings and cathedrals. In America, churches have survived, however, by adapting to secularization and by commending themselves in terms that are attractive to "secular man."
This strategy has insured short-term success, but it also has meant that the church has undergone many fundamental changes. In its organization, for example, the church tends to take on the characteristics and values of contemporary secular organizations and institutions; it comes to resemble surrounding technological and bureaucratic organizations. Too frequently such churches are beehives of committee meetings, questionnaires, self-studies, memos and related paraphernalia that primarily serve to maintain institutional momentum.
Further, the secular church alters traditional ministries and multiplies a host of new ministries through which it seeks to "meet needs." Committed to meeting consumer demands, the accommodating church must check constantly the latest shifts and fads which announce the appearance of a new "need." In all of this the secular church almost comes to resemble a delicatessen offering samples to attract customers. Its members are exhorted to be involved, to share, and occasionally, to serve. Far less frequently are they called to serious Bible study and discipleship.
Traditional tasks of the minister are increasingly unimportant, both to society and within the secular church. In order to survive within the organizational framework of the emerging church, many ministers find themselves forced to pay close attention to style and technique. They must learn how to meet the demands of an increasingly secular clientele whose expectations frequently are shaped more by TV personalities than by Christian belief. Ministers succeeding within the secular church thus may come to know more about managerial techniques, executive decision making, group dynamics and popular psychology than they know about the Bible.
The importance of the church also changes its meaning in the life of the average member of the secular church. While for older members the church may retain something of its traditional importance, for many younger and newer members it no longer offers a clear body of beliefs and values vital for life. Rather, the outlook of the average member of the emerging church tends to be an unexamined and unstable hodgepodge of bits and pieces taken from folk wisdom, Dear Abby, horoscopes and the latest version of psychotherapy.
Not surprisingly, the church tends to play the same marginal role in the life of the average member as it does in the secular society. While the church may be a beehive of activity, church life is leisure time pursuit. The secular church becomes a "Christ Club," providing social contacts, entertainment and recreation (and just possibly minimal religious obligation to salve consciences).
Clearly, in spite of its many apparent successes, the secular church pays a terribly high price for its survival. For example, it may be just a matter of time until many members, having their appetites for "need gratification" whetted by the church, turn to such alternatives as health spas and dinner clubs.
Behind the many problems of the secular church, however, lies a fundamental problem: there is little place for the God of the Bible in the secular church. In a secular world that must quantify, manage, program and manipulate, the unfathomable mystery and majesty of the biblical God simply does not fit. The so-called transcendence of the secular church is a false transcendence which merely uses the Creator in a thinly veiled adoration of and infatuation with the creature.
Worship, for example, in the secular church tends to become a quasi-entertaining media event that must "meet my needs" by providing an emotional outlet and offering an occasion for conviviality. Worship is fundamentally not an occasion marked by reverence, awe and an awareness of the mysterious otherness of the God of Abraham and Isaac, Jesus and Paul. Salvation likewise comes to be loosely linked with the gratification of personal needs and the pursuit of "self-realization."
While all of this largely goes unnoticed in easy times (and one suspects that the secular church is only possible in comfortable times), leaders of the secular church find hard going when it becomes necessary to call for humility, patience, or self-sacrifice. There can be very little place for the cross in the easy and self-centered life of the "Christ Club."
Recovering the way is always a difficult task. For the secular church, recovery will depend upon a painfully honest recognition of the problem. It will demand nothing short of a recovery of transcendence, an openness to the biblical record of God's shattering and unpredictable incursions into human history. This, in turn, will rekindle near-forgotten memories that "His ways are not our ways." It will remind us that God is worshipped not because he is useful but because he is God. Only such a turn to the God above our countless false gods can free us from our self-imprisonment within a web of illusions and fantasies.
Ironically, only with a recovery of the biblical faith can we make any meaningful and lasting attempt to "meet needs." For it is only within a recovery of the biblical perspective that we may recognize and redirect false needs and identify secondary needs idolatrously masquerading as ultimate ones.
Consequently, repentance also will be a necessary part of recovering the way. We will confess that we have put ourselves in the place of God. We will recognize that it is not our tasks to manage, much less manipulate, the church; nor are we called to be executives in the Kingdom of God --only servants. We are not called to be successful; we are called only to be faithful. -- Michael R. Weed, associate professor at the Institute for Christian Studies, Austin Texas. Christian Chronicle, June 1985. (Editor's Note: Without question, the Church of Christ (all segments) is following the other denominations in fast becoming "a secular church," adapting to the self-centered culture. The Church of Christ is now an institutional, corporate operation based upon business and worldy concepts. The church is no longer merely God's people; it is an organization.)


text:
Audi Rs5 185052 850.34 KB
"Audi Rs5 185052" is an awesome wallpapers presented here by Becky Smith at the top resolution of 2560x1440px and size of 850.34 KB. More of such great image you always can find at the Cars category or with our "Related walls" widget. Upload date - 16 June 2015
Popular Wallpapers


text:
- Research
- Open Access
The important ergot alkaloid intermediate chanoclavine-I produced in the yeast Saccharomyces cerevisiae by the combined action of EasC and EasE from Aspergillus japonicus
© Nielsen et al. 2014
- Received: 14 April 2014
- Accepted: 24 June 2014
- Published: 12 August 2014
Ergot alkaloids are a group of highly bioactive molecules produced by a number of filamentous fungi. These compounds have been intensely studied for decades, mainly due to their deleterious effects in contaminated food and feeds, but also for their beneficial pharmaceutical and agricultural applications. Biosynthesis of ergot alkaloids goes via the common intermediate chanoclavine-I, and studies of the key enzymes, EasE and EasC, involved in chanoclavine-I formation, have relied on gene complementation in fungi, whereas further characterization has been hampered by difficulties of poor EasE protein expression. In order to facilitate the study of ergot alkaloids, and eventually move towards commercial production, the early steps of the biosynthetic pathway were reconstituted in the unicellular yeast Saccharomyces cerevisiae.
The genomic sequence from an ergot alkaloid producer, Aspergillus japonicus, was used to predict the protein encoding sequences of the early ergot alkaloid pathway genes. These were cloned and expressed in yeast, resulting in de novo production of the common intermediate chanoclavine-I. This allowed further characterization of EasE and EasC, and we were able to demonstrate how the N-terminal ER targeting signal of EasE is crucial for activity in yeast. A putative, peroxisomal targeting signal found in EasC was shown to be nonessential. Overexpression of host genes pdi1 or ero1, associated with disulphide bond formation and the ER protein folding machinery, was shown to increase chanoclavine-I production in yeast. This was also the case when overexpressing host fad1, known to be involved in co-factor generation.
A thorough understanding of the enzymatic steps involved in ergot alkaloid formation is essential for commercial production and exploitation of this potent compound class. We show here that EasE and EasC are both necessary and sufficient for the production of chanoclavine-I in yeast, and we provide important new information about the involvement of ER and protein folding for proper functional expression of EasE. Moreover, by reconstructing the chanoclavine-I biosynthetic pathway in yeast we demonstrate the advantage and potential of this host, not only as a convenient model system, but also as an alternative cell factory for ergot alkaloid production.
- Ergot alkaloids
- Chanoclavine-I
- Pathway reconstruction
- S. cerevisiae
- Metabolic engineering
Ergot alkaloids (EA) belong to a diverse group of natural compounds with a range of biological activities that have important applications in medicine and agriculture [1-3]. Some of these compounds have a notorious neurological effect on humans, possibly due to the structural similarity of these compounds to neurotransmitters like serotonin and dopamine [4]. EAs are produced by a variety of plant associated fungi, mainly of the genera Claviceps and Aspergillus. The production of ergot alkaloids has been linked to biosynthetic gene clusters found in several species, first in Claviceps purpurea [5] and later in Aspergillus fumigatus [6,7], Neotyphodium lolii [8], and others (see [3] for review).
The first step of the common pathway is the electrophilic aromatic addition of dimethylallyl-pyrophosphate (DMAPP) to the 4 position of tryptophan to form dimethylallyl-tryptophan (DMAT). The reaction is catalysed by a prenyl transferase, DmaW (fgaPT2 in A. fumigatus) [6,9]. The second step, the methylation of DMAT to form N-methyl-4-dimethylallyltryptophan (Me-DMAT), is catalysed by the methyltransferase EasF (fgaMT in A. fumigatus) [10].
While the biosynthesis of Me-DMAT is well understood, the mechanistic basis behind the conversion of Me-DMAT to chanoclavine-I is less clear. The conversion of Me-DMAT to chanoclavine-I was investigated by gene disruption and complementation studies in C. pupurea and A. fumigatus: Lorenz and co-workers [11] used a mutated C. purpurea strain P1 to show that deletion of the easE_Cp (ccsA) gene abolished production of chanoclavine-I and any downstream products. Instead an accumulation of Me-DMAT was seen, indicating a block in the pathway after this intermediate. Alkaloid biosynthesis could be restored by expressing a GFP fusion construct of the easE_Cp gene. Analogously, Goetz and co-workers [12] disrupted the easC_Af gene in A. fumigatus, and also observed accumulation of Me-DMAT and the absence of downstream products. Furthermore, a similar pattern was observed when easE_Af was disrupted, in concordance with the C. purpurea results (above). In both of the easE and easC deletion strains, the alkaloid pathway could be restored by re-introduction of the corresponding wild type allele. Most recently Ryan and co-workers [13] transferred part of the A. fumigatus EA cluster, comprising the four genes dmaW, easF, easC, and easE, into A. nidulans, a fungus known as a non-producer of any EA. This partial cluster conferred the ability to produce chanoclavine-I, further suggesting that EasE and EasC are sufficient for the conversion from Me-DMAT. However, the involvement of enzymes from cryptic EA clusters in A. nidulans cannot be excluded [14].
Interestingly, EasC_Af contains a C-terminal amino acid motif (SRL), which is a classic type 1 peroxisomal targeting signal (PTS1) [15]. Inspection of related published sequences reveals that similar PTS1 signals are found in many EasC homologues from Aspergillus spp., but not from Claviceps spp. EasE_Af, on the other hand, appears to have an N-terminal signal peptide for the secretory pathway, as previously suggested [12]. The sequence has the typical core of hydrophobic amino acids within the first 15–30 residues, and a signal peptide is predicted by the SignalP model [16]. However, the implication of these localisation signals remains unclear, particularly in light of the apparent co-operation between EasC and EasE. The EasC proteins have similarity to peroxisomal catalases [6,7,17]. EasC_Af (the easC_Af gene product) was purified after expression in E. coli, and in vitro catalase activity of EasC_Af was shown using H2O2 as substrate [12]. However, when the enzyme was incubated with Me-DMAT, no new product was detected. Extensive efforts were made to produce active EasE_Af from E. coli or S. cerevisiae [12]. However, in all cases, incubation of EasE with Me-DMAT, or of EasE with Me-DMAT and EasC, failed to produce any new product. Therefore, biochemical studies to understand the transformation of Me-DMAT to chanoclavine-I have not been possible.
As an alternative approach to gain information about the early EA pathway we undertook the reconstitution of the chanoclavine-I pathway in yeast (S. cerevisiae). Yeast is the work horse of eukaryotic gene expression and is easily amenable to genetic manipulation [18,19]. Heterologous genes and proteins are generally well expressed, and yeast has a long history of use for industrial production of a variety of products [20,21]. We report here the successful engineering of yeast for de novo production of chanoclavine-I, overcoming the previously reported difficulties of expressing EasE. Further, we provide new insight into the roles of EasC and EasE in the intriguing biochemical conversion from Me-DMAT into chanoclavine-I, the key step in the early ergot alkaloid pathway.
Prediction of dmaW coding sequences
An ergot alkaloid gene cluster was recently identified in A. japonicus [22], one of several fungal species currently being investigated for their capacity to produce molecules of potential commercial importance [23-25]. A. japonicus was previously reported to produce cycloclavine [26], an EA of the clavine group, and the genome sequence of the cluster displays high homology to EA clusters of Claviceps spp. and Aspergillus spp. This cluster therefore provided an interesting alternative for studying the chanoclavine-I pathway. The putative coding sequences (CDS) of dmaW, easF, easC, and easE were predicted, using free online gene prediction software and by alignment to homologues in the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Analysis of the dmaW sequence in the A. japonicus genome revealed two different CDS predictions: one prediction was for a single open reading frame of 1602 bps, corresponding to a 534 amino acid protein, whereas another prediction, with the same translation start codon, was for two exons of 1287 bps and 150 bps separated by an 85 bps intron, corresponding to a 479 amino acid protein. Both of these predictions were slightly longer than similar DmaW enzymes found in the GenBank database, where the lengths of DmaW homologues were in the range of 435–465 amino acids. Moreover, a multiple protein alignment in GenBank, using either of the two A. japonicus DmaW predictions as query, showed a lack of homology beyond approx. 428 amino acids, i.e. beyond the predicted first exon. A somewhat similar situation was seen for the two entries of A. fumigatus DmaW (Additional file 1: Figure S1). Hence, we decided to test, not only the two predictions encoding 534 amino acids (dmaW_Aj1) and 479 amino acids (dmaW_Aj2), but also a shorter version encoding 429 amino acids (dmaW_Aj3), corresponding to the predicted first exon. For easF, representing the next step in the pathway, we tested the homologues easF_Af and easF_Aj, from A. fumigatus and A. japonicus, respectively.
Me-DMAT production in yeast
Genes used in this study
CDS name
Amino acids
easC_Aj -PTS1
easE_Aj -N sig.
Chanoclavine-I production in yeast
Peroxisomal targeting signal is not needed in yeast
N-terminal signal is crucial for EasE function
A common feature observed for EasE enzymes is a high number of hydrophobic amino acids at the N-terminal end, which is typically associated with signal peptides for the secretory pathway. Analysis of the amino acid sequence of EasE_Aj, using the online SignalP server [16], confirmed this notion by predicting a signal peptide. A putative cleavage site in EasE_Aj was predicted after pos. 29, i.e. between A and V. We used this information to prepare a truncated version of the enzyme, which lacked the N-terminal sequence, and expressed the corresponding gene from the pRS313-G/C plasmid, in the Me-DMAT producing strain, together with easC_Aj (in pRS315-P/A). The N-truncation seriously impaired the functional expression of EasE_Aj, and essentially no chanoclavine-I was detected using this enzyme. Instead, accumulation of the precursor compound Me-DMAT was observed (Figure 4).
Pdi1 or Ero1 overexpression improves the activity of EasE in yeast
Fad1 overexpression improves the activity of EasE in yeast
EasE has previously been described as a flavin adenine dinucleotide (FAD) dependent reductase or dehydrogenase, and in support of this the structural similarity to the class of berberine bridge enzymes (BBE) has previously been pointed out [11]. In plants the BBE enzyme is involved in alkaloid biosynthesis, and it has been associated with transport from ER to the vacuole [29]. BBE was shown to bi-covalently bind a flavin co-factor [30] and the binding site, involving a histidine and a cysteine, seems to be conserved in EasE_Aj and other EasE enzymes (Additional file 1: Figure S4). With ample evidence that EasE is FAD dependent, we speculated whether an increased supply of this co-factor would directly improve the activity of EasE. An attempt to increase the supply via the growth medium showed no effect on chanoclavine-I production (data not shown), so instead we cloned the native yeast fad1 gene, which encodes the enzyme responsible for synthesis of FAD from flavin mono nucleotide (FMN). Over-expression of this gene, in a strain harbouring the chanoclavine-I pathway, led to a 2.5 fold increase in chanoclavine-I production (Figure 6) which we interpreted as an effect of improved co-factor supply.
Heterologous expression in yeast of the four enzymes DmaW_Aj2, EasF_Af, EasC_Aj, and EasE_Aj led to production of chanoclavine-I, demonstrating that yeast is a suitable host for producing ergot alkaloids. This work also confirms, in accordance with previous published studies, that EasC and EasE are solely responsible for the conversion of Me-DMAT to chanoclavine-I without the involvement of other EA enzymes. Secondary metabolism in S. cerevisiae is quite limited and it seems unlikely that any yeast enzyme would be involved in the highly specialized metabolic process of ergot alkaloid biosynthesis. The production of chanoclavine-I depended on the expression of EasE from A. japonicus, whereas EasE from A. fumigatus or C. purpurea were not active when expressed in yeast. We speculate that the predicted coding regions of easE_Af and easE_Cp, along with dmaW_Aj3, could be incorrect (see below, and Additional file 1: Figure S3 and S5). Certainly, the prediction of intron and exon sequences in filamentous fungi is still a challenge.
Our results support the notion that EasE and EasC co-operate to produce chanoclavine-I, but it is still not clear how or where this process occurs. Knockout experiments of easC and easE in Aspergillus fumigatus [12], along with the data presented here, indicate that the catalytic activities of two classes of enzymes, a catalase encoded by easC and a FAD-dependent oxidoreductase encoded by easE, are both required to convert Me-DMAT to chanoclavine-I. Deletion mutants of easC and easE accumulate Me-DMAT, which suggests, though does not prove, that these two enzymes act in a coordinated fashion. It is also possible that the ergot alkaloid pathway requires disproportionation of H2O2, and that the function of EasC is to remove H2O2 derived from the oxidative activities of EasE. More extensive biochemical experiments with active EasE enzyme would be able to shed further light on these questions.
As shown here the putative PTS1 signal of EasC_Aj was not essential for function, and it is possible that even with the PTS1 signal the natural cellular distribution involves multiple locations, as seen for some native yeast proteins [31,32]. The N-terminal signal of the EasE, on the other hand, was clearly crucial for function, and the fact that a native yeast signal peptide from Pdi1 partially restored function indicates that EasE contains a genuine ER targeting signal. This would suggest that folding and disulphide bond formation in the ER is needed for proper maturation of EasE and that it passes through the secretory pathway before, possibly, joining up with EasC. Cellular localization studies might help elucidate this puzzle, just as further progress toward obtaining in vitro activity of EasC and EasE should allow us to explore the interdependence of these two enzymes on one another.
The case for oxidative folding of EasE in ER is supported by the observed increase in chanoclavine-I production after over-expression of either of the two key enzymes in the yeast disulphide bridge formation machinery. Ero1 is a sulfhydryl oxidase responsible for generating disulphide bonds that are passed on to Pdi1, which in turn oxidizes the cysteines of newly translated proteins [28,33,34]. The EasE family contains several highly conserved cysteines that would be available for oxidation, although one cysteine is likely to be involved in binding the FAD co-factor.
Interestingly, a multiple sequence alignment of the EasE_Aj and its closest homologues showed some unexpected dissimilarity regarding EasE_Af and EasE_Cp (Additional file 1: Figure S5). The first approx. 130 amino acids of EasE_Af showed no similarity to the consensus sequence, whereas EasE_Cp seemed to be completely lacking the N-terminal domain. An earlier prediction of EasE_Cp [11] suggested a 483 amino acid peptide derived from two exons. A newer GenBank entry [GenBank:JN186799], however, predicts a third exon at the 5-end and encodes a 595 amino acid protein, including a signal peptide as predicted by the SignalP model [16]. The study [11] reported complementation of an easE_Cp gene knock-out after integration of a PCR fragment comprising the same easE gene including its native promoter region. We speculate that this PCR fragment may by chance have included the first exon, allowing correct splicing and, hence, full complementation. Complementation by re-integration into the easE locus was excluded, since integration at the niaD locus was confirmed by Southern hybridization. A similar approach was used for studying the easE gene in A. fumigatus [12]. The native easE gene was first disrupted, which abolished function, and the WT sequence was then re-integrated along with 1445 bps 5'-flanking sequence. The inclusion of upstream sequence would allow the fungus to splice the gene correctly, and any divergence between the mature mRNA and the predicted CDS of easE_Af would not have been detected. We analysed the genomic region of A. fumigatus chromosome II using an online intron prediction model, which suggested an 1809 bps easE_Af coding sequence, encoding a protein of 602 amino acids including a putative N-terminal signal peptide. This newly predicted protein shows very high sequence similarity to other EasE proteins in the multiple alignment (Additional file 1: Figure S3). We synthesized a yeast codon optimized version of this predicted easE_Af, and tested its ability to support chanoclavine-I formation. In a strain co-expressing dmaW_Cp, easF_Af, and easC_Aj, the expression of either easE_Af or easE_Aj resulted in similar levels of chanoclavine-I production (data not shown). This would indicate that, at least in this case, the EasC and EasE enzymes from different fungi are capable of working together. However, further studies are needed to confirm the results, and to investigate the nature of co-operation between EasC and EasE.
We show here that EasC and EasE are responsible for the conversion of Me-DMAT to chanoclavine-I in yeast. Although the activity of these enzymes appears to be linked, the cellular localization of the enzymatic reaction is unclear. The N-terminal peptide sequence of EasE was identified as a signal for the secretory pathway, and a role for ER-associated folding was supported by a beneficial effect of overexpressing host enzymes involved in protein disulphide bond formation.
Surprisingly, a putative C-terminal PTS1 in EasC appeared to be non-essential, at least in a yeast host background, and the cellular localization of EasC cannot be predicted. Further studies will be needed to address questions regarding cellular localization of the enzymes, and much work is still pending towards a full understanding of the biochemical reactions involved. Nonetheless, the knowledge we have obtained in this study should enable further elucidation of these reactions.
Importantly, we also demonstrate that the biosynthetic pathway for chanoclavine-I, the central biosynthetic precursor for all ergot alkaloids, can be transferred to the industrially important host, S. cerevisae. This discovery will greatly facilitate further genetic and metabolic engineering of the ergot alkaloid pathway, which will potentially have a broad range of pharmaceutical and agrochemical applications. Our results therefore provide an entry point on the road to commercial scale production of known or novel ergot alkaloids with important industrial applications.
DNA and protein sequence analysis
Computer-aided sequence analysis was done using Vector NTI 9.1.0 software (Invitrogen Corp. 2004) and the free online software FGENESH (http://linux1.softberry.com/berry.phtml), GENSCAN (http://genes.mit.edu/GENSCAN.html), and the NCBI server (http://www.ncbi.nlm.nih.gov). Signal peptides were predicted using the SignalP 4.0 tool [16].
Preparation and cloning of genes in yeast expression vectors
Synthetic genes, codon optimized for expression in yeast, were manufactured by DNA2.0 Inc., Menlo Park, CA, USA or GeneArt AG, Regensburg, Germany. All sequences were derived from A. japonicus, A. fumigatus, or C. purpurea. The genes encode the amino acid sequences, plus a translation stop codon, of DmaW_Af [GenBank: XP_756141], DmaW_Cp [GenBank:CAB39314], EasF_Af [GenBank:XP756143], EasC_Af [GenBank:XP756140], EasE_Af [GenBank:XP756142], and EasE_Cp [GenBank:CAB39328]. The sequences dmaW_Aj1, easF_Aj, easC_Aj, and easE_Aj were predicted, based on the genomic DNA sequence of the cycloclavine gene cluster of A. japonicus [22]. All genes were synthesized with the DNA sequence AAGCTTAAA, containing a HindIII restriction recognition site, at the 5'-end and with a SacII recognition site at the 3'-end (CCGCGG), and these sites were used for cloning. All PCR primers used for sub-cloning contained these sequences. Standard PCR conditions were used according to manufacturer's recommendations (BioRad iProof High Fidelity DNA polymerase, Cat. #172-5302).
Gene dmaW_Aj3 was prepared by PCR using dmaW_Aj1 as template, and dmaW_Aj2 was prepared by sequential extension PCR, using dmaW_Aj3 as template, thus adding the second exon in two steps (Additional file 1: Table S1). The easC_Aj version without C-terminal PTS1 signal was prepared by PCR amplification of the coding sequence (CDS) without the 9 bps before the stop codon. The EasE_Aj N-terminal truncation (EasE_Aj -N sig.) was done by PCR, amplifying the corresponding CDS without the first 87 bps. The forward PCR primer inserted an alternative ATG translation start site. The fusion of an N-terminal signal peptide from Pdi1 to the N-truncated EasE_Aj was done by overlapping extension PCR, fusing the two amplicons to encode Pdi1-EasE_Aj (Additional file 1: Table S2). The CDS of the native yeast genes pdi1 [GenBank:D00842], ero1 [GenBank:NM_001182493], and fad1 [GenBank:NM_001180104] were amplified from genomic DNA by PCR.
For expression, all genes were cloned into expression vectors based on pRS313, pRS315, and pRS316 [35]. These vectors had been provided with a new multi-cloning site (MCS) linker, inserted between the two PvuII sites. The basic design of the MCS was SrfI-AscI-BglII-HindIII-SfiI(a)-SfiI(b)-SacII-SphI-AscI-SrfI [36]. The linker allowed cloning of promoter sequences into BglII and HindIII, and terminators into SacII and SphI restriction sites to create yeast expression cassettes. Promoters and terminators were amplified from yeast genomic DNA by PCR (Additional file 1: Table S3) for preparing three expression cassettes containing 1) a GPD1 promoter and a CYC1 terminator (G/C), 2) a PGK1 promoter and an ADH2 terminator (P/A), and 3) a CUP1 promoter and an ADH1 terminator (C/A). The new expression vectors were named pRS31X-G/C, pRS31X-P/A, and pRS31X-C/A, where X designates 3, 5, or 6. All genes used in this study (Table 1) were cloned into expression cassettes of these vectors.
Construction and integration of yeast gene expression cassettes
Constructs for integration were prepared for the integration sites YORWΔ22 and YPRCΔ15 [37], and cloned in unique EcoRI and HindIII sites of a pUC19 vector backbone. The homologous regions were constructed from two PCR fragments, which were then combined by overlapping extension PCR. The PCR primers introduced the restriction sites AscI and NotI between the two original fragments, and SbfI sites at the outer ends. The KanMX cassette, flanked by loxP sites, was excised from pUG6 [38] and inserted into NotI. Two expression cassettes (described above) were inserted into the AscI site. The first cassette was amplified by PCR, changing one AscI site to a MluI. After cloning this fragment, the second cassette was inserted into the single regenerated AscI site. The entire construct was released by SbfI and used for integration. This approach was used to first integrate dmaW_Aj2 (G/C cassette) and easF_Af (P/A cassette) into the yeast genome at the YORWΔ22 site. After excision of the KanMX marker [38], the easC_Aj (G/C cassette) and easE_Aj (P/A) cassette were integrated into the YPRCΔ15 site. Yeast transformations were done using the LiAc method [39].
Yeast strain and culture conditions
Chanoclavine-I production was achieved in several S. cerevisiae strain backgrounds, including S288C, W303, and others (not shown). The strain used to prepare the results in this report had the genotype MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0. Engineered yeast strains were grown in standard SC broth with 2% glucose, minus leucine and histidine (ForMedium, Hunstanton, U.K.). When appropriate, CuSO4 was added to a final concentration of 300 μM for induction of gene expression. Cultures for analysis were started from re-streaked, single colonies. These were grown overnight in standard SC broth, and then diluted to an optical density, at 600 nm, of 0.1 to start the main culture. Main cultures were grown at 30°C with constant shaking at 150 rpm, 5 cm amplitude, for 72 hours in 250 ml shake flasks containing 25 ml medium. Final optical density was measured at 600 nm, typically reaching 12–14, although small variations were seen between individual experiments.
Analytical procedures
For analysis yeast cultures were spun down for 10 min at 1000 × g. The pellet and the supernatant were separated. Without further purification, 5 μl of supernatant were injected in a UPLC-TOF (Waters Acquity™ Ultra Performance LC, Waters, Milford, Mass., USA) coupled to a micrOTOF-Q II (Bruker Daltonik GmbH, Bremen, Germany). Stationary phase was an Acquity UPLC® Bridged Ethyl Hybrid (BEH) C18, 1.7 μm, 2.1 × 100 mm column. Liquid chromatography used mobile phases of H2O + 0.1% formic acid (A), and acetonitrile + 0.1% formic acid (B), in a linear gradient of 1% to 100% B in 5 min. The column was washed for 1 min in 100% B, and then equilibrated for 1.5 min in 1% B. Detection of compounds was done by a photo diode array using the following parameters: λ range 210 nm to 500 nm. Resolution 1.2 nm. Sampling rate 5 points/s. ELSD parameters: gain 50, gas pressure 40 psi, nebulizer mode heating, power level 80%, drift tube 80°C. TOF parameters Source: End Plate Offset −500 V. Capillary −4500 V. Nebulizer 1.6 bar. Dry gas 8.0 l/min. Dry temperature 180°C. TOF parameters Scan mode: MS Scan. Mass range from 80 to 1000 m/z. Quantification of chanoclavine-I was based on in-house, purified reference compound.
Preparative procedures
For compound purifications yeast cultures were spun down for 10 min at 1000 × g. The supernatant was adjusted to pH = 10 with 10 M NaOH and extracted by liquid/liquid extraction with an equal volume of ethyl acetate. The crude extract was dried under vacuum and reconstituted with dimethyl sulfoxide (DMSO) to a concentration of 100 mg/ml and then purified on a preparative HPLC system (Waters, Milford, Mass, USA). Stationary phase was an XBridge™ preparative C18, 5 μm, 19 × 250 mm column. Liquid chromatography used mobile phases of H2O + 0.1% trifluoroacetic acid (A), and acetonitrile + 0.1% trifluoroacetic acid (B), in a linear gradient of 1% to 30% B in 40 min. The column was washed for 5 min in 100% B, and then equilibrated for 5 min in 1% B. Fractions were collected every 2 min and analyzed as above. Fractions containing the purified analyte were pooled and dried under vacuum.
The authors wish to thank Federico Brianza for advice on the chemical analysis, Jens Klein for solving the compound structures (NMR), and Donna Williams and Louise Hunter for technical support.
Authors' Affiliations
- Schardl CL, Panaccione DG, Tudzynski P: Ergot alkaloids - biology and molecular biology. Alkaloids Chem Biol 2006, 63:45–86.View ArticleGoogle Scholar
- Haarmann TY, Rolke Y, Giesbert S, Tudzynski P: Ergot: from witchcraft to biotechnology. Mol Plant Pathol 2009, 10:563–577.View ArticleGoogle Scholar
- Wallwey C, Li SM: Ergot alkaloids: structure diversity, biosynthetic gene clusters and functional proof of biosynthetic genes. Nat Prod Rep 2011, 28:496–510.View ArticleGoogle Scholar
- Tudzynski P, Correia T, Keller U: Biotechnology and genetics of ergot alkaloids. Appl Microbiol Biotechnol 2001, 57:593–605.View ArticleGoogle Scholar
- Tudzynski P, Hölter K, Correia T, Arntz C, Grammel N, Keller U: Evidence for an ergot alkaloid gene cluster in Claviceps purpurea . Mol Gen Genet 1999, 261:133–141.Google Scholar
- Unsöld IA, Li SM: Overproduction, purification and characterization of FgaPT2, a dimethylallyltryptophan synthase from Aspergillus fumigatus . Microbiology 2005, 151:1499–1505.Google Scholar
- Coyle CM, Panaccione DG: An ergot alkaloid biosynthesis gene and clustered hypothetical genes from Aspergillus fumigatus . Appl Environ Microbiol 2005, 71:3112–3118.Google Scholar
- Fleetwood DJ, Scott B, Lane GA, Tanaka A, Johnson RD: A complex ergovaline gene cluster in epichloe endophytes of grasses. Appl Environ Microbiol 2007, 73:2571–2579.Google Scholar
- Tsai HF, Wang H, Gebler JC, Poulter CD, Schardl CL: The Claviceps purpurea gene encoding dimethylallyltryptophan synthase, the committed step for ergot alkaloid biosynthesis. Biochem Biophys Res Commun 1995, 216:119–125.Google Scholar
- Rigbers O, Li SM: Ergot alkaloid biosynthesis in Aspergillus fumigatus. Overproduction and biochemical characterization of a 4-dimethylallyltryptophan N-methyltransferase. J Biol Chem 2008, 283:26859–26868.View ArticleGoogle Scholar
- Lorenz N, Olsovská J, Sulc M, Tudzynski P: Alkaloid cluster gene ccsA of the ergot fungus Claviceps purpurea encodes chanoclavine I synthase, a flavin adenine dinucleotide-containing oxidoreductase mediating the transformation of N-methyl-dimethylallyltryptophan to chanoclavine I. Appl Environ Microbiol 2010, 76:1822–1830.Google Scholar
- Goetz KE, Coyle CM, Cheng JZ, O'Connor SE, Panaccione DG: Ergot cluster-encoded catalase is required for synthesis of chanoclavine-I in Aspergillus fumigatus . Curr Genet 2011, 57:201–211.Google Scholar
- Ryan KL, Moore CT, Panaccione DG: Partial reconstruction of the ergot alkaloid pathway by heterologous gene expression in Aspergillus nidulans . Toxins 2013, 22:445–455.Google Scholar
- Inglis DO, Binkley J, Skrzypek MS, Arnaud MB, Cerqueira GC, Shah P, Wymore F, Wortman JR, Sherlock G: Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of Aspergillus nidulans, A. fumigatus, A. niger and A. oryzae. BMC Microbiol 2013, 13:91.View ArticleGoogle Scholar
- Thoms S, Debelyy MO, Connerth M, Daum G, Erdmann R: The putative Saccharomyces cerevisiae hydrolase Ldh1p is localized to lipid droplets. Eukaryot Cell 2011, 10:770–775.Google Scholar
- Petersen TN, Brunak S, von Heijne G, Nielsen H: SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 2011, 8:785–786.View ArticleGoogle Scholar
- Haarmann T, Machado C, Lübbe Y, Correia T, Schardl CL, Panaccione DG, Tudzynski P: The ergot alkaloid gene cluster in Claviceps purpurea : extension of the cluster sequence and intra species evolution. Phytochemistry 2005, 66:1312–1320.Google Scholar
- Nevoigt E: Progress in metabolic engineering of Saccharomyces cerevisiae . Microbiol Mol Biol Rev 2008, 72:379–412.Google Scholar
- Chen Y, Daviet L, Schalk M, Siewers V, Nielsen J: Establishing a platform cell factory through engineering of yeast acetyl-CoA metabolism. Metab Eng 2013, 15:48–54.View ArticleGoogle Scholar
- Borodina I, Nielsen J: Advances in metabolic engineering of yeast Saccharomyces cerevisiae for production of chemicals. Biotechnol J 2014, 9:609–20.Google Scholar
- Mattanovich D, Sauer M, Gasser B: Yeast biotechnology: teaching the old dog new tricks. Microb Cell Fact 2014, 13:34.View ArticleGoogle Scholar
- Schröder H, Hoff B: Gene Cluster for Biosynthesis of Cycloclavine. US Patent Application. Geneva, Switzerland: WIPO; 2012. WO 2012/116935 A2, September 2012.Google Scholar
- Facchini FD, Vici AC, Benassi VM, Freitas LA, Reis RA, Jorge JA, Terenzi HF, Polizeli Mde L: Optimization of fibrolytic enzyme production by Aspergillus japonicus C03 with potential application in ruminant feed and their effects on tropical forages hydrolysis. Bioprocess Biosyst Eng 2011, 34:1027–1038.Google Scholar
- Pradeep S, Faseela P, Josh MK, Balachandran S, Devi RS, Benjamin S: Fungal biodegradation of phthalate plasticizer in situ . Biodegradation 2013, 24:257–267.Google Scholar
- Bel-Rhlid R, Pagé-Zoerkler N, Fumeaux R, Ho-Dac T, Chuat JY, Sauvageat JL, Raab T: Hydrolysis of chicoric and caftaric acids with esterases and Lactobacillus johnsonii in Vitro and in a gastrointestinal model. J Agric Food Chem 2012, 60:9236–9241.Google Scholar
- Furuta T, Koike M, Abe M: Isolation of Cycloclavine from the Culture Broth of Aspergillus japonicus SAITO. Agric Biol Chem 1982, 46:1921–1922.Google Scholar
- Delic M, Valli M, Graf AB, Pfeffer M, Mattanovich D, Gasser B: The secretory pathway: exploring yeast diversity. FEMS Microbiol Rev 2013, 37:872–914.View ArticleGoogle Scholar
- Gasser B, Saloheimo M, Rinas U, Dragosits M, Rodríguez-Carmona E, Baumann K, Giuliani M, Parrilli E, Branduardi P, Lang C, Porro D, Ferrer P, Tutino ML, Mattanovich D, Villaverde A: Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microb Cell Fact 2008, 7:11.View ArticleGoogle Scholar
- Alcantara J, Bird DA, Franceschi VR, Facchini PJ: Sanguinarine biosynthesis is associated with the endoplasmic reticulum in cultured opium poppy cells after elicitor treatment. Plant Physiol 2005, 138:173–183.View ArticleGoogle Scholar
- Winkler A, Motz K, Riedl S, Puhl M, Macheroux P, Gruber K: Structural and mechanistic studies reveal the functional role of bicovalent flavinylation in berberine bridge enzyme. J Biol Chem 2009, 284:19993–20001.View ArticleGoogle Scholar
- Petrova VY, Drescher D, Kujumdzieva AV, Schmitt MJ: Dual targeting of yeast catalase A to peroxisomes and mitochondria. Biochem J 2004, 380:393–400.View ArticleGoogle Scholar
- Lu Q, McAlister-Henn L: Peroxisomal localization and function of NADP + −specific isocitrate dehydrogenases in yeast. Arch Biochem Biophys 2010, 493:125–134.View ArticleGoogle Scholar
- Kramer G, Boehringer D, Ban N, Bukau B: The ribosome as a platform for co-translational processing, folding and targeting of newly synthesized proteins. Nat Struct Mol Biol 2009, 16:589–597.View ArticleGoogle Scholar
- Feige MJ, Hendershot LM: Disulfide bonds in ER protein folding and homeostasis. Curr Opin Cell Biol 2011, 23:167–175.View ArticleGoogle Scholar
- Sikorski RS, Hieter P: A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae . Genetics 1989, 122:19–27.Google Scholar
- Naesby M, Nielsen SV, Nielsen CA, Green T, Tange TO, Simón E, Knechtle P, Hansson A, Schwab MS, Titiz O, Folly C, Archila RE, Maver M, van Sint Fiet S, Boussemghoune T, Janes M, Kumar AS, Sonkar SP, Mitra PP, Benjamin VA, Korrapati N, Suman I, Hansen EH, Thybo T, Goldsmith N, Sorensen AS: Yeast artificial chromosomes employed for random assembly of biosynthetic pathways and production of diverse compounds in Saccharomyces cerevisiae. Microb Cell Fact 2009, 8:45.View ArticleGoogle Scholar
- Flagfeldt DB, Siewers V, Huang L, Nielsen J: Characterization of chromosomal integration sites for heterologous gene expression in Saccharomyces cerevisiae . Yeast 2009, 26:545–551.Google Scholar
- Güldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH: A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 1996, 24:2519–2524.View ArticleGoogle Scholar
- Gietz RD, Schiestl RH: High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2007, 2:31–34.View ArticleGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.


text:
Congratulations, you're in a modern band. In addition to the basic vox, guitar, drums, bass, you've got yourself a keyboard player. Or maybe you're using a laptop. Or maybe you've got a sampler or an iPod, or anything else that lacks the standard 3-pin XLR output. You've probably heard about a DI or a Direct Box, and how it might make these devices sound better or perform better during a live performance.
So, do you need one?


text:
Momento Mori VI by Riza Peker depicts the paradoxical coexistence of tropical botanicals and a macabre skull. Curioos acrylic glass prints provide images with crystal clear clarity and added depth. Images are printed on a high-quality, HD premium photo paper and are mounted on the back of clear acrylic substrate. The high gloss of the acrylic enhances the hues and vibrancy of the image, providing for intense, rich colors. All prints are signed and sent with a certificate of authenticity.
Acrylic glass print. Momento Mori VI by Riza Peker. Available in size Small 16" x 20", Medium 20" x 25" or Large 28" x 35".
Details: Archival quality print mounted on the back of a 1/4" thick, clear acrylic substrate. Polished edges. Includes wall hanging hardware. Manually numbered, signed and shipped with a certificate of authenticity. Limited edition. Exclusive to Curioos.
AHAlife is committed to help you live a conscious and inspired life.
Many of the items you discover here are exclusive to AHAlife, which is what makes our community of designers and artisans so special.
If you find one of our products anywhere else for less than it's being sold on our site, we will match it.
Our dedicated Support Team is here to help. Reach out any time at:


text:
September 1, 2019 - May 31, 2020
Location: Schools throughout the NW region
School Assemblies
Our assembly program brings the historical journey of taiko from Japan to the United States. This 45-50 minute presentation intersperses background information with exciting taiko demonstrations and dynamic hands-on student participation. Contact Young Audiences of Oregon and SW Washington to book a taiko performance for your school.
Korekara Taiko can provide several choices of residencies to engage your students in specially designed taiko programs by grade and focus. In all residencies, each student will be actively engaged in taiko activities and learn important lessons with direct instruction from passionate and experienced taiko professional teachers/artists. Within the taiko session students will actively participate in individual and ensemble taiko exercises. Length of each class session will vary depending on the grade level.


text:
Lawyers file 753rd appeal to visit Kurdish leader Öcalan
Abdullah Öcalan's lawyers filed an application with the Bursa Prosecutor's Office for the 753rd time to visit their client. Since July 27, 2011, each application has been rejected.
Kurdish leader Abdullah Öcalan's lawyers İbrahim Bilmez, Faik Özgür Erol and Fuat Coşacak today filed an appeal with the Bursa Prosecutor's Office for the 753rd time to visit their client.
Previous appeals by the Öcalan's lawyers have been rejected regularly by the Bursa Chief Public Prosecutor's Office on grounds of "Restrictions against convicts" listed in the Execution of Penalties and Security Measures Act No.5275.
The appeals have been rejected to date, citing excuses like "bad weather", "broken boat", "boat repairs" and "State of Emergency".
The Kurdish People's Leader Abdullah Öcalan has been denied any legal aid for more than six and a half years. He is subject to total isolation on the prison island of Imrali in the Sea of Marmara.


text:
Interestingly, the patent relates to Internet Protocol (IP) communication systems and, more particularly, to broadband telephony services having "distributed local gateways". Don't all broadband telephony services use "distributed local gateways"? After all, you need distributed local gateways to help get cheaper rates. So Does this mean Packet8 can go after Vonage, AT&T, Time Warner, and all the other providers of broadband telephony for patent infringement? Patent
I'll have to contact my sourcs at 8x8 and find out exactly what this patent entails. Stay tuned...
Relatedly, since its establishment in 1987, 8x8 has been awarded seventy-three (73) United States patents covering a variety of voice and video communications, processing and storage technologies.
Also, Packet8 is up 1.5% and Vonage is down 5.99%. Coincidence?


text:
The Wave
- 400 Metacom Avenue, Bristol, RI 02809
- Bristol County
- (401) 253-8811
Great food. Great drinks. Great times.
All efforts have been made to ensure the accuracy of the information on this website, however it is subject to change. Information is updated in an ongoing manner in partnership with local tourism offices, individual businesses and organizations and via a direct feed from goprovidence.com and discovernewport.org
What's Nearby 400 Metacom Avenue, Bristol, RI 02809


text:
We don't charge more for our friendly office staff and our knowledgeable trusted advisers! Plus we are always here to take your call.
Our installation teams are energetic, friendly, and respectful. When we do the installations, we will walk the job site before you turn it over to the customer, to make sure everything works properly. Because that's just what we do.
You can browse our suppliers for the product you want, or you can let us help you decide which product will work best for your new construction project, matching the architectural style, color, and quality.
Contact a Great Northern Trusted Advisor to get started. Quotes are Free!


text:
Since yesterday we've had 25cm with up to 30cm expected by Sunday!Opening day is November 27 so get your gear ready and stay tuned to whistlerblackcomb.com to find out what's open. Canadian & Washington State residents don't miss your last chance to get this season's best EDGE Card deal. Ski or ride from $59 a day plus get bonus offers and perks. If you haven't got your EDGE Card now's the time to buy, early bird pricing ends this Friday, November 21st. Check out whistlerblackcomb.com/deal to purchase or call 1-800-766-0449.Check out our Season 2 Episode 1 of the Whistler Blackcomb Snowcast, presented by TELUS. You can view it at whistlerblackcomb.com/podcast
NEW SNOW, EDGE Cards and Podcasts
published by WB


text:
NEST® Views Construction and Facilities Maintenance Management through Multiple Lenses:
Your construction and facilities maintenance management program has more to offer than well-maintained interiors and exteriors. In addition to preserving your brand, a top-notch program has the ability to directly impact your bottom line. NEST's strategic approach delivers:
- A Comprehensive Strategic Plan that analyzes current operations, desired states, current budgets and process, and recommends best practices that will help you achieve desired results.
- Business Analytics Reporting that evaluates and reviews current performance against KPIs and industry standards.
- Accurate Financial Forecasting and Reporting that delivers real-time data on your current spend, including accruals forecasted to match your fiscal budgets and reporting requirements.
- Asset Capture and Reporting that tracks facilities assets and captures expenses to calculate the true life of an asset, while providing critical data for ROI calculations, warranty management and total value.
- Customized Reporting that can be delivered via automated subscription, housed real-time on your dashboard, and customized by audience and data needs.
The leader in multi-facility maintenance and construction management, NEST® specializes in consulting, technology and financial solutions, all backed by world-class customer service.
- Proven Multi-Facilities Advisor. With 20+ years' experience in financial acumen and unique cost reduction strategies, we understand how to deliver unparalleled programs for maximum ROI.
- Multi-Facility Consulting. Using our expertise, we orchestrate strategic programs in which services, frequency, product, process and cost yields significant value across your brand.
- Service Provider Network. Our ISP model engages the expertise and accountability of local providers who take pride in their work and are a part of your community.
- Cutting Edge Technology. Our proprietary in-house technology solution enables customized, real-time management and troubleshooting of the entire multi-location work order process while providing intuitive financial modules, business analytics, asset management tools and KPIs.
- Command Center. A seasoned operations team of long-term NEST employees manages, executes and tracks performance of your construction and facilities maintenance management operations and budget. We are a true 24/7 environment and no division of our staff is outsourced.
The NEST Method™ is a three-pronged approach to multi-facility management that helps you build, manage, execute and track your construction and facilities maintenance management business plans and budgets to achieve your desired goals: enhanced programs, maximum value, reduced risk and excellent customer satisfaction. This industry-leading solution is executed by our seasoned team of operations and customer service experts trained to optimize your business plans and budgets while providing the best value over time through the only customer loyalty program in the industry.
NEST Consult™
- Seasoned strategic consultants
- Fully optimized on-time, on-budget programs
- Improved customer experience
NEST ISP® Network
- Expansive vetted community of local independent providers invested in your outcome
- Consistent, high-quality customer experience
- Dependable service, with performance continuously managed, executed and tracked
NEST Facilitate™
- Cutting edge technology platform customized to your needs and requirements
- Seamless management of work orders, ISPs and data flow
- Fully integrated financial modules for long-term financial planning
- Dashboard functionality to measure and track KPIs


text:
Websites Hosted in ns30.domaincontrol.com Nameserver
The Nameserver ns30.domaincontrol.com have a IP Address of 126.96.36.199 which is hosted on United States.
Outside The Beltway | OTB | Online Journal of Politics and Foreign Affairs
Analysis of U.S. politics, world affairs, and public policy from an educated, thoughtful perspective
Commentary Magazine
Conservative American journal of politics, Judaism, social and cultural issues.
Kurzweil Accelerating Intelligence
Launched in 2001, KurzweilAI explores the forecasts and insights on accelerating change articulated in Ray Kurzweil's landmark books — notably The Age of Spiritual Machines and...
PRADO PHP Framework
Center for Responsible Lending
Helping American families build wealth by promoting fair financial practices. We offer research, policy and legal solutions, and consumer tips.
XMind: Professional & Powerful Mind Mapping Software
The Blonde Salad
The official website of The Blonde Salad: post, photo, video by Chiara Ferragni.
43 Rumors
State Bar of Arizona :: Protecting the Public While Advancing the Legal...
The State Bar of Arizona serves the public and enhances the legal profession. AZBar helps consumers search for and find lawyers.
Binary Option Platforms | Binary Options Broker Reviews
Binary Options Platform Reviews, Read and compare between various binary option platforms. Leave comments and discuss particular platform. Learn the pros & cons of each...
Pro Sound Web
ProSoundWeb.com Pro Audio Web News, Forums & Info: Live Sound Reinforcement, Recording Studio, Pro Sound Audio Forums, Audio Industry News
160by2 - Free SMS to Any Mobile in India, 100% Free to SMS
Free SMS, Send Free SMS, Send Free SMS to india, Free SMS in india, Free SMS Sites, free SMS to Mobiles, Free SMS website, Free SMS website in india, Free SMS service, Free SMS...
iPhone Hacks | iPad, iPod Touch, iPhone Blog
iPhone Hacks - The #1 iOS Blog for the latest iPhone, iPad and iPod Touch Hacks, Apps, Jailbreaks, News, Rumors, Games, Reviews, Tweaks, Tips, Tricks, How-To Guides
Kikolani - Blog Marketing and Blogging Tips for Professional Bloggers
Kikolani offers blog marketing and blogging tips for personal, professional, and business bloggers. Other topics include SEO and social media marketing.
Odd Future Talk | Official Blog and Message Board for OFWGKTA
Job search starts here | Recruit.net
Search jobs at Recruit.net. Search thousands of job sites, newspapers and company career pages with one click.
Seattle Met
Smart. Authoritative. Entertaining. With a bold design, eye-catching photography, and an editorial voice that's at once witty and in-the-know, Seattle Metropolitan is our city's...
WHSR | Best Web Hosting Reviews & Web Development Guide
Unbiased web host reviews - Find honest and in-depth hosting reviews by experienced webmasters based on real usage experience.


text:
Walk or cycle through heathland and forest by Morden Bog National Nature Reserve, one of the largest valley mire habitats in England.
There are picnic benches and a signposted walk. Cyclists are welcome on 2 metre or wider tracks. Horse riders by permit only. The Sika Trail (a 7 mile family cycle trail) and Woodlark Trail (2 mile walking trail) are accessed from the Sika Trail car park off the Bere Road or via the Northport Greenway.
The Northport Greenway provides a safe and pleasant walking and cycling route into the Forest from Wareham Quay via Wareham Railway Station.
Guide Prices
|Ticket Type
|Ticket Tariff
|Free Admission
Note: Prices are a guide only and may change on a daily basis.
Map & Directions
Public Transport Directions
ARRIVING BY TRAIN: The nearest railway station is at Wareham (on the London Waterloo to Weymouth line). It is a half mile walk or cycle along the Northport Greenway into the Forest. You can take your bike on the train.
ARRIVING BY BUS: Wareham Railway Station bus stop. From Bournemouth take the M1 or M2 to Poole, then transfer to number 40 or X54. From Poole take the number 40 or X54. From Swanage take the number 30 or 40. From Weymouth take the number 30 or X54. It is a half mile walk or cycle along the Northport Greenway into the Forest.
JOURNEY PLANNING: To plan your journey contact Traveline on 0871 200 2233 or visit their website: www.travelinesw.com to look up bus timetables.
- Picnic Site
Establishment Features
- Dogs Accepted - Dogs to be kept on a short lead in some areas of the forest during bird nesting season (February to August).
- Open All Year
Event Facilities
- Free Entry
Key Features
- Forest Location
- In countryside
- Outdoor Attraction
Parking & Transport
- On site parking - for cars. No charge.


text:
Bathroom Taps: Which ones to choose
These times' toilet add-ons are becoming significant not just for comfort but also for providing a look that was beautiful additionally. Numerous kinds of toilet accessories, today are offered in the market; few of these are just in the form of bath faucets. You will find amounts of bath faucets. Bunch of the things are there-in the marketplace and you also just need selecting the one which fits with all the insides of your own bathroom. There are faucets like Electric Showers Energy Showers, Miscellaneous Showers, Fixed Shower-Head Packages and a lot more.
Different selections can be made by you for various kinds of toilet faucets that could not be unsuitable for your toilet accessories substitutions. You'll get many fashions that changes in the contemporary fashions to fashions that are conventional. You will find people who favour their toilet collection that is traditional with faucets that are contemporary and in the ending they've got a fashion that is unique. You can even pick faucets that have been in line with perspectives and styles in the bath. These faucets and an important part perform for offering a general effect on the mindset of your bath. For offering a sophisticate prognosis for their bath, people searching should choose water faucets that are high-priced.
The toilet faucets are discovered in various kinds of substances. Really there are numerous kinds of faucets like ceramic faucets, stainless and opera steel faucets and many more. So, you'll discover other kinds of faucets that will be in coatings that are distinct. Actually a faucet which match the decoration and color scheme together with the bath room can be selected by you. It's possible for you to choose also the forms of faucets that often enhance the problems that are ceramic nicely for the toilet. These will create how that prognosis on the entire be to offer the exceptional appearance within your toilet to you. For instance, bathing tubs, bowls that are well-rounded seem along with faucets having spouts that are ring-shaped. A lot of people consider the baths, railings, closet, in truth water faucets and and towel as well as Showerhead Packages are among the add-ons which unite them all collectively.
Another thought that is really important is installing the toilet faucets. That is owing to the fact bulk of the faucets may be repaired on tub or the sink. A few of these may also be set behind the sink as well as around the partitions. The areas where the water faucets are to repaired have become significant because it could determine dimensions as well as the style that you need to get. For meeting your requirements, you'll discover numerous kinds of faucets. Handle faucets are the kind which can be utilized readily plus they're not unhygienic additionally. They will be got by you in angles that are curved, right and prevent. Cold and comfortable water can be controlled by handle faucets of solitary kind. On the course where they may be set that determines the heat of the swimming. Screw faucets that are down is purely normal form of faucets which are normally offered in bulk houses. They're having a water faucet with which offer of cold and warm water may be guaranteed. It's possible for you to choose from several faucets.


text:
Report from Congressman Gerry Connolly
8th Congressional District Report
10th Congressional District Report
Supreme Court Decision Trumps People's Will
What Democrats Have Accomplished
FCDC Denounces Virginia Attorney General Opinion
Rex Simmons, FCDC Chair
This is the first edition of The Democrat in an entirely electronic format. The Democrat should be an informative newsletter with timely information about the Fairfax County Democratic Committee; insights on political issues in our community, the commonwealth, and the nation; and status reports from Democratic candidates and their campaigns that will help us get them elected and keep them in office.
You will notice some added articles and commentary in this month's edition. In addition, there are some of the reports you have been familiar with from our past printed editions. For now, the format will be simple because it can be produced at a lower cost and facilitate easier navigation through the document.
I would like to see The Democrat continuously improve. I have appointed a temporary Communications Task Force, chaired by Tom Goldsmith from Hunter Mill, to recommend further improvements. I welcome any suggestions you have, and you can send them directly to me or to Tom. Also, I am asking for a volunteer who would be Editor-in-Chief and who will help carry out this vision. My email address is email@example.com and Tom's email is email@example.com.
One of the most important events for FCDC this year is our annual Jefferson Jackson Dinner on Sunday evening, May 23 at the Tysons Corner Hilton. Please plan to attend. For information and reservations, go to www.fairfaxdemocrats.org.
Report from Congressman Gerry Connolly
Election Day is only 6 months away and it is critical that we mobilize, energize, and educate our Fairfax Democrats now so that we have strong turnout at the polls on November 2nd.
Six months seems like a long time, but it will be here before we know it. Midterm elections always result in a substantially lower turnout than presidential election years so it is important that we redouble our efforts this year. I assure you that this election with be anything but a cake walk.
I appreciate the hard work and planning that Chair Rex Simmons, the Steering Committee, and many of you on FCDC are already putting into the process.
Over the last 14 months in the U.S. House of Representatives, I have accomplished much of what I pledged to do during the 2008 campaign despite Republican obstructionism and misleading rhetoric that has scared many voters.
We have turned the corner on the worst recession since the Great Depression. Here in Fairfax, my efforts in Congress have helped save the jobs of hundreds of teachers, kept class sizes down, and helped county government minimize the impact of the sluggish economy on our children. I have brought hundreds of millions of dollars in transportation funds into the county for Rail to Dulles, completion of the Fairfax County Parkway, the Fair Lakes Interchange, and other regional transportation priorities.
I supported our successful effort in the House to take concrete steps to reduce our dependence on foreign oil and promote renewable energy sources and green jobs. I voted to stand with middle-class taxpayers in Northern Virginia by enacting real Wall Street reform that holds these elite investment banks accountable for their actions and I championed the new law that clamps down on the credit card companies and their abuse of consumers. I was also proud to support passage of the largest single tax cut in history for small businesses and 95 percent of all Northern Virginians.
Most recently, we enacted meaningful health care reform that will prohibit insurance companies from dropping people when they get sick, allow parents to keep their kids on their insurance until they are 26, make it illegal for insurance companies to deny coverage to those with pre-existing conditions, and strengthen Medicare and close the prescription drug donut hole for our seniors. Meanwhile, the two Republicans running to face me in November want to cater to the big insurance companies and repeal these meaningful reforms.
After Republicans cavalierly squandered record surpluses and left us with record deficits, we are slowly but surely putting our economy back on track. But we still have work to do and we have to take more steps to reduce the deficit. That is why I voted for the PAYGO law and other deficit reduction measures to rein in deficit spending.
Between now and November, we have to remind the voters that it was the Republicans who got us into this economic mess. And we must fight rigorously for every vote because we cannot afford to turn Congress back over to the Republicans. They will undo everything we have done to improve our economy, worship at the altar of big oil, big banks, and the insurance companies, weaken Social Security and Medicare, and return our health care system to a path that is not sustainable.
We can't let them do it, and together we will make sure they won't.
8th Congressional District Report
Pixie Bell, 8th CD First Vice Chair
The 8th CD met on April 19 and voted to buy ½ page in each 8th CD jurisdictions J.J. dinner programs. As most of you know, the State Democratic Party has vacancies in the office of Vice Chair for Finance and 1st Vice Chair for Organization. The elections will be held at the June meeting, with filing deadline June 4, before 5 PM. Congressman Moran attended the 8th meeting. Charley Conrad gave us a quote from Jim "We have a lot to be proud of, a good record to run on, a dynamic President who is delivering on his campaign promises and we are digging ourselves out of an enormous debt." Jim also announced that a growing number of insurance companies are voluntarily providing coverage for graduating college seniors under the age of 26 in advance of the September start date that is in the recently signed health insurance reform legislation. He noted that Republicans in Congress who wish to repeal the health bill ought to be clear just what that will mean for these young adults. In case anyone missed it, Jim is now Chairman of the Interior Appropriations Subcommittee and Vice Chair of the Defense Appropriations Subcommittee. See below pictures from a luncheon with Vicki Kennedy, and a dedication of Our Military Kids Program, securing $800,000 for fiscal year 2010 and supporting their request for $2.5 million this year, and Jim receiving an award from Everybody Wins!
Excerpt from Remarks by Congressman Jim Moran (VA-08) at Our Military Kids Awards Ceremony, April 2010
"The United States military has had to rely heavily on the Guard and Reserve to fight the two wars in Afghanistan and Iraq, which has been ongoing since 2001 and 2003 respectively. While the families of the active duty soldiers live on or near military installations, the same cannot be said for many National Guard and Reserve families."
"Our Military Kids provides grants for after-school activities for the children of Guard and Reserve service members who don't live close to a military installation and therefore most pay out-of-pocket for sports, tutoring, and other recreational and educational activities. I'm proud to support this meaningful service."
Moran has been a dedicated champion of the Our Military Kids program in Congress, securing $800,000 for fiscal year 2010 and supporting the organization's request for $2.5 million this year.
Congressman Moran received a Congressional Service Award from Everybody Wins, the DC reading mentoring program last month. The award was presented by the late Sen. Ted Kennedy's wife Vickie. Rep. Moran's staff has seven people reading in Everybody Wins' power lunch reading program. Staffers spend one hour a week during their lunch break reading to children in the DC public schools.
10th Congressional District Report
Patsy Brown, 10th CD Vice Chair, Sam Crockett 10th CD Chair
Previously, the Tenth District Democratic Committee (10 CD) choose to hold a Primary as our method of nomination. However, only one person was certified as a candidate on the April 14th deadline, so the Primary was cancelled.
Our candidate is JEFF BARNETT of McLean.
Barnett's Campaign Manager, Marc Abanto reports that Jeff has already set up two campaign offices: one is Herndon and one in McLean—with others to follow across the district.
On April 24, the 10 CD held its Spring Fling fundraiser at Dondoric Farm in The Plains, which is a historic home owned by one of our 10 CD members, Bob Zwick.
We bid adieu to long-time FCDC member and 10 CD member, Eileen Manning, who is moving to Florida. We will miss Eileen's energy.
The next 10 CD meeting will be in the Barnett for Congress Herndon Office @ 2 pm on Sunday, May 23, 2010; after we adjourn, several of us will attend the FCDC JJ Dinner.
Virginia's 10th Congressional District includes all or portions of 10 jurisdictions: all of Clarke, Frederick, Loudoun, and Warren Counties, and the Cities of Manassas, Manassas Park, and Winchester; and portions of Fairfax, Fauquier, and Prince William Counties.
Supreme Court Decision Trumps People's Will
A commentary by Sandra J. Klassen and Dan Walsch, National Affairs Committee Co-chairs
The Citizens United v. FEC Supreme Court decision trumps the will of the people. In rendering their decision, the majority in Citizens United v. FEC swept away decades of concerns expressed regarding corporate influence on US elections. From Brandeis to Rhenquist to Marshall, justices have consistently, and at times unanimously, ruled that the interests of the public in maintaining fair and open elections extended to limiting corporate campaign expenditures. It cannot be stressed enough how complete a reversal this ruling represents.
Corporations may now spend freely on elections. Not only were the strictures of McCain-Feingold lifted, no new legislation attempting to implement any similar ordnance is likely to survive a court challenge. The only corrective measure that could possibly return corporations to their status as instruments of law and not as legal persons would be a constitutional amendment, and the odds of such an amendment passing with the necessary three-quarters of the states ratifying it are remote.
The consequences of this ruling on federal elections are cause for concern. However, it is the effect on the Supreme Court's modus operandi that is equally chilling. In 1936, Justice Brandeis stressed the importance of following the rules the Court had developed for its own governance, noting that making rulings on broad constitutional theories rather than narrow statutory grounds would create greater risk for error. Few rulings have been as broad as the majority's ruling in Citizens United. For example, in 1986, Sandra Day O'Connor ruled with the majority upholding the anti-sodomy statutes in Bowers v Hardwick, which was subsequently overturned by Lawrence v Texas, in which O'Connor also concurred. This might appear as "flip-flopping". However, as O'Connor noted in her concurring decision in Lawrence, the question in Bowers was one of due process while the question in Lawrence was one of equal protection. O'Connor, despite identifying the legal and moral problem with Bowers knew her hands were tied by how the Supreme Court limited its rulings. She was only able to correct the injustice 17 years later.
The majority did not just change 100 years of legal precedent, but also upset 220 years of Court precedent by choosing to move beyond the complaint filed by Citizens United and consider the First Amendment implications of the entire statute. This established a precedent for future courts to make dramatic rulings that even alters the balance of powers. Whereas before the Court could only go so far in contradicting the legislation passed by our elected members of congress and signed by our elected president. In effect, the Court has taken the power to trump the will of the people in toto.
The only bright spot in this ruling is that eight members of the court concurred on the issue of transparency. Congress may require ads disclose who is paying for them, and those regulations enforcing transparency might mitigate their influence. Senator Chuck Schumer and Congressman Chris Van Hollen have started drafting legislation to those ends, and supporting that legislation is the most important thing we can do until a future court can find some way of overturning Citizens United.
What Democrats Have Accomplished
A commentary by Rex Simmons, FCDC Chair
Despite all the histrionics from the other side of the aisle, President Obama and Congressional Democrats have accomplished a lot. As grassroots activists we should arm ourselves with a few facts to counter arguments we read and hear. Here's a primer.
Providence District puts on a going away party for two fantastic long time members who are moving out of Providence District. Patty Parker (left) and Colleen Leyrer (right), with Chair Carrie Nixon (center)
Sully Report
Dana Cimino, Sully Chair and Stu Bromberg, Member
A well attended Sully Democratic Committee meeting took place on Tuesday, April 6. The main focus was long term support of FCDC and our candidates in the fall elections. We are already planning Back to School Nights, the preparation of personalized literature supporting President Obama's programs and ramping up a letter writing campaign for neighborhood and local newspapers. Mark Abanto, Campaign Manager, and Dan Henshaw, Field Coordinator for Jeff Barnett's 10th Congressional District Campaign brought us up to date on office openings, events and fundraisers. We also received a report on events and additional information on the 10th Congressional race; updates from the FCDC meeting including the hiring of two organizers and were reminded that there will be a retreat for precinct captains on May 16th. Please become an active member and join us for our next meeting. Your participation is essential to the success of the group and our candidates. Additional information may be found at our site www.sullydemocrats.org
Lee Report
Hugh Robertson and Steve Bunn, Lee Co-Chairs
Thanks to everyone who came out to our Opening Day fundraiser on April 10th. We had a good turnout, lots of food, and fun in the sun including our own baseball game (later in the day). Also thanks to our host, Carson Evans, and our Sponsors, Bernard Fagelson and Michael Wells.
Our next meeting will be on Thursday, May 27th at 7:30pm in the home of Susie Warner at 6229 Edison Road. We hope you will join us as we talk about our upcoming Fairfax JJ Dinner and plan for our Precinct parties.
Braddock Report
Jan Hedetniemi and Lynn Miller, Braddock Co-Chairs
Braddock District's signature event is Derby Day and we welcome everyone to celebrate with us on Saturday, May 1st from 4:00 to 6:30 p.m. at Kilroy's Restaurant. Join your fellow Democrats and our elected officials including newly elected Senator David Marsden and Delegate Eileen Filler-Corn! Auction items will be plentiful as will food including shrimp and desserts. The big screen television set on Kilroy's back porch is a great way to watch the Derby and to cheer on your pick.
Click here for details of all events below
May 1st
Mt. Vernon Garden Party
Braddock Derby Day
May 2nd
Providence MayFest
May 4th
Springfield District Meeting
Sully District Meeting
May 5th
Drinking Liberally – Mt.Vernon/Lorton
May 6th
Bylaws Taskforce Meeting
May 8th
Voter Registration Kick-off Rally
Cinco De Surovell Fundraiser
May 10th
Sports in the City
May 11th
FCDC Steering Meeting
May 12th
Mason District Meeting
May 13th
Education Committee Meeting
May 16th
Precinct Captain Retreat
Potomac Nationals Game with Springfield Dems
3rd Annual Friends of Foust Fundraiser
Democratic Women of Clifton Family Picnic
May 17th
8th CD Meeting
11th CD Meeting
May 18th
Mt. Vernon District Meeting
May 19th
National Affairs Committee Meeting
May 20th
Hunter Mill District Meeting
May 23rd
FCDC Jefferson-Jackson Dinner
May 24th
Precinct Ops. Committee Meeting
May 25th
FCDC General Committee Meeting
May 26th
State and Local Affairs Committee Meeting
May 27th
Lee District Meeting
Voter Registration Committee Meeting
May 30th
Viva Vienna Festival
May 31st
Viva Vienna Festival
The Democrat is a monthly email publication of the Fairfax County Democratic Committee. The purpose of The Democrat is to provide information to Committee members and other interested persons through district and committee reports, campaign reports, articles, and fliers. Views expressed do not necessarily represent those of the FCDC unless specifically approved by an appropriate committee resolution.
Generally material submitted to The Democrat should be limited to 300 words unless exceptions are made by the Editor or FCDC Chair. Please email material to email@example.com. Attach text file or photos, or copy the material into the email message. Deadline for submission is the third Monday of each month by 5:00 pm for inclusion in the following month's issue.
One-page fliers advertising Democratic fundraisers and events will be included with The Democrat if submitted by the deadline. Limited numbers of The Democrat are mailed, primarily to those who do not have access to email. FCDC charges $35 for each flier from non-FCDC organizations.
Contact FCDC Executive Director Mike Burns at firstname.lastname@example.com with any questions about how to submit material or fliers.


text:
Picture: Arctic Monkeys frontman Alex Turner and drummer Matt Helders come through the arrivals hall at LAX Airport Los Angeles, California - 12.02.12
Are Arctic Monkeys playing the London 2012 Olympics' Opening Ceremony tomorrow (July 27, 2012)? An image tweeted by Argentinian journalist Martin Rinaldi certainly suggests that may be the case. The photo appears to show the band's front man Alex Turner, performing live. The caption 'Not For Transmission' partly obscures his face and it's not entirely clear whether or not it was a live shot or not.
Even if Alex was onstage rehearsing with the band, there's no news as to whether the band is playing the opening ceremony or the closer. The stadium will be in constant use until the end of the games so it's feasible that acts playing the closing ceremony could be practising now. However, last month (June 2012), a document was leaked that reportedly revealed that the Sheffield band were indeed scheduled to play, along with M.I.A. and Adele. Paul McCartney also let slip that he would be performing as part of the Danny Boyle-curated opening ceremony.
Last month, Boyle revealed a few elements of the opening ceremony, which reportedly cost in the region of 27 million. Some of these include a "mosh pit" at each end of the arena and a model of Glastonbury Tor, in a nod to the Glastonbury Festival taking a year off this year. The music line-up though, is supposed to have ben kept a secret, though it might well be worth your while betting on Arctic Monkeys putting in an appearance.


text:
Michael J. Barrett is certified by the New Jersey Supreme Court as a Certified Civil Trial Attorney. He is a member of the firm's Personal Injury Team and primarily represents clients in product liability, medical malpractice, negligence and commercial litigation matters. Mr. Barrett has represented the New Jersey Turnpike Authority, the Middlesex County Improvement Authority, Roosevelt Care Center and the Middlesex County Utilities Authority in litigation matters. He served as the president of the Middlesex County Trial Lawyers Association from 1997-1998. He has also served as a Trustee of the Trial Attorneys of New Jersey from 1994 through 1997. Mr. Barrett is also a contributing author in a text entitled "New Jersey Product Liability Law," New Jersey Law Journal Books, (1994), and the author of "New Jersey's Wrongful Death Law: A Matter of Economics," 245 N.J. Law Journal 38 (April 2007). He is a member of the American Association for Justice, the New Jersey State Bar Association, The Middlesex County Bar Association, the Middlesex County Trial Lawyers Association, and regularly serves as a bar panelist for the Middlesex County Bar Panel program.
Areas of Emphasis: Negligence, Automobile, Industrial Accidents, Medical Malpractice, Product Liability, Commercial matters


text:
The Expressions had a fantastic competition weekend in Chesterton, Indiana winning Grand Champion at the 3rd Annual Trojan Championship. Hosted by the Chesterton High School Trojans, the competition was as fierce as the weather was blustery. Due to the snow and a forecast of blizzard like conditions, several schools left early and others were complete no shows.
The Expressions' day started off with the solo competition at 2:30 p.m. Five talented singers competed including Cole Festenstein, Domingo Morales, Milan Babic, Jeremy Hamburger and Mia Connery. Festenstein impressed the judges and placed second in the men's competition. The first place male winner left early, giving Festenstein the opportunity to wow the audience before the finals with his performance of "Flight."
At 5:10 p.m. The Expressions' Tribute to Michael Jackson rocked the preliminary competition. Despite a minor lighting glitch, the show went on and what a "Thriller" night it was. The Expressions singing and dancing outperformed the competition and helped them to win Best Large Showchoir, Best Vocals, Best Choreography, Best Band and Best Visuals. The audience loved the moonwalk, the Jackson Five and the grand finale with the special glow in the dark effects. Rachel VanderVeen was named Outstanding Performer by the judges.
The Trojan Championship finals were organized a little bit different than other championships. Typically only the large show choirs compete in the finals, but at this championship the six groups with the highest scores were the ones invited to compete. This provided a nice mix of large choirs and unisex groups for the audience to watch, as well as a mix of traditional and more contemporary shows. As one of only five groups invited to compete in the finals, The Expressions once again impressed the audience and judges with their singing, dancing and high energy performance. At the end of the night and the end of a suspenseful awards program, The Expressions were named Grand Champion.
The Expressions have several more performances coming up this season, including:
- Chicagoland Showcase at John Hersey High School March 7th.
- Wheaton-Warrenville South Competition at Wheaton-Warrenville South High School March 14th.
- Show Choir National – Nashville Competition at The Grand Ole Opry in Nashville, TN March 26-29th.
We hope to see you at one of them.