text:
Italo Calvino said: The more enlightened our houses are, the more their walls ooze ghosts. Describe the ghosts that live in this house: Image credit: "love Don't live here anymore…" – © 2009 Robb North – made available under Attribution 2.0 Generic
8 of us all in line waiting for our final time to leave these doors and be a family once more. A tragedy took us, hunger we shouldn't have felt, on bended knees in prayer we knelt. 6 of us sons and daughters. Mommy and Daddy passed while we slumbered. One remains that we must wait for before we are all together once more.


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Release Year: 2016
Genres: black girls, breast torture, Brunettes, Busty, ethnic, pantyhose, sponge gag
Video language: English
This clip starts where Part 1 ended… Sassy's captor takes the rope that had secured her waist to the post and now uses it to secure her waist to the chair. As is her gag wasn't strapped in tight enough, It gets shoved in her mouth further when he starts wrapping elastic vetwrap around it. He doesn't stop there though… he continues wrapping her head with it until the entire roll is used up. He now ropes up each of her ankles to the sides of the chair. He leaves her for a bit, but finally returns with some thin twine. He uses it to tightly bind Sassy's tits. Of course he's going to slap and squeeze them now, who could resist? He removes her blindfold and gag. You can tell it was tight by the deep gag strap marks on her cheeks. He now shoves a huge sponge in her mouth that is about the same size as her head. It's secured by many wraps of clear tape. Sassy's captor leaves, but returns with a thin wooden paddle. He clasps one hand over her gagged mouth and then starts smacking her bound tits with the paddle. In between the tit smacking he smacks her pussy with it as well. After what seems like an eternity of tit and pussy slapping, the man leaves her to endure her bondage and aching ass and tits. This is where Part 2 ends.
Format: mp4
Duration: 18:43
Video: 1280×720, AVC (H.264), 2907kbps
Audio: 124kbps

subset: commoncrawl


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Are you considering writing the copy for your own Website? While writing your own copy is certainly a very tempting way to save money, cutting corners on the development of your Web content is a serious mistake that can lead to a reduction in Web traffic while also tarnishing the reputation of your business or organization.
Anyone Can Do It, Right?
When it comes to writing Web content, some business owners mistakenly believe it is something they can easily do. After all, everyone has taken writing courses in high school and many people have taken a composition class or two in college. It can't be that hard, right?
The reality is that writing for the Web is quite different from the type of writing most people have completed in high school and in college. Not only does Web content need to be interesting and engaging to the reader, it also needs to incorporate keywords that will help attract search engine spiders.
Simply put, thinking you can write your own Web content because "everyone knows how to do it" is the same as saying you can be a professional singer because everyone knows how to sing. Sure, you may know some of the fundamentals, but this doesn't mean your skill is refined to the point that you can get the type of reaction you want from your audience.
Those Keywords Can Be Tricky
Speaking of keywords, incorporating them into your Web content can be far trickier than you might think. Sure, it doesn't take much thought to throw some keywords into a sentence here and there, but including keywords in a way that flows seemingly within the content is a far more difficult endeavor. Ideally, your site visitor shouldn't even be able to identify which words are the keywords. While even a professional writer may not be able to achieve this goal with keywords that are particularly difficult, a writer with keyword writing experience will be able to create content that is engaging and meaningful to the reader while also incorporating the keywords that have been selected for the site.
Your Reputation is on the Line
Finally, it is important to keep in mind that your reputation is on the line each and every time someone visits your Website. While your average visitor won't be able to look at your site and know how it was programmed and designed, he will be able to read every last word that has been published on your site. Therefore, you want to make certain those words are sending the right message. Do you really want to trust your reputation to content that is sub-par?
The bottom line is that hiring a professional to create the content for your Website can be far less expensive that you might think. In addition, given all of the benefits that hiring a professional provides, it is certainly a worthwhile expense.


text:
Land Your Dream Job Without Applying Online
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1 Comment
A good resume objective is the difference between landing the interview and having your application tossed in the trash.
Here's why:
You've probably heard that employers spend an average of six seconds scanning your resume. But do you know where they spend the majority of that time?
Scanning the resume objective.
It's the easiest way for them to qualify (or disqualify) candidates, which is why it's critical that you structure your objective in a way that's easy to digest and hooks your potential employer from the first bullet.
This, my friend, is easier said than done. If you did a Google search for "resume objective" you probably came across a bunch of conflicting advice that left you wondering:
At least that's how I felt when I was looking for help during my job search. This article aims to change that. It's going to be your one stop shop for crafting a resume objective that achieves a single goal: getting you the interview and the job offer.getting you the interview and the job offer.
Here's what we're going to cover:
Once you're done with this article, go ahead and check out this guide on writing resumes that will actually get you hired (the first time).
Your resume objective is the first section that appears below your name and contact information on your resume:
A highly effective objective includes 3-5 bullets of the most relevant experience and compelling results when compared to the job description.
Other career websites and "gurus" may advocate for different formats. Since founding Cultivated Culture, I've had thousands of resumes come across my desk which has given me the opportunity to test each style, collect data, and monitor success. The bullet-style that I'll be walking you through in this article outperformed every other format.
It's specifically designed to hook the reader, highlight the information they are using to qualify candidates, and deliver it in a format that is easy to digest. Here's a quick comparison of the bullet-style vs. a paragraph style resume objective (in the context of an outbound sales role):
Example of Bullet Style
Example of Paragraph Style
At [Company], I am responsible for managing a team of six sales people that have a track record of consistently over-attaining against quota and drove ~40% of net new sales last year for the company. Most recently, I restructured our sales pitch process to incorporate a "Challenger Sale" mindset from the initialpoint of contact through to the close. It helped us increase our sell through rate by 15% and increased our average deal size by 29% versus the previous process. Finally, diversity and inclusion is a huge passion of mine. I created a group called embRACE which aims to help make diversityand inclusions a core pillar of our company's culture.
As it turns out, the psychological drive is much more powerful than the physical experience of withdrawal. Cocaine, which also produces little withdrawal sickness but does create extreme craving, was once seen as nonaddictive — that was before America was introduced to crack in the 1980s. More than a century ago, Mark Twain summed up the essence of the problem, in reference to the addictiveness of tobacco: "Giving up smoking is easy," he said. "I've done it thousands of times."
I personally had the same experience with heroin. I got through the nausea and chills at least five different times, but avoiding the psychological draw of the drug long-term was much harder than suffering through a few weeks of withdrawal symptoms. Quitting cocaine is similar. ( More on Time.com: Best of 'Stoner Cinema' ).
So the question is, how does marijuana compare to these classically addictive drugs? Estimates vary, but compared with tobacco, which hooks about 20% to 30% of smokers, marijuana is much less addictive, coming in at 9% to 10%. In contrast, 23% to 25% of heroin users get addicted, along with 15% of alcohol users and 15% to 20% of those who use cocaine.
Marijuana is the most heavily used drug in the country — by their 20s, 56% of Americans have tried it — but
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of people who are in addiction treatment report that marijuana is their primary drug. In contrast, just 2% of young adults have ever tried heroin, but heroin addicts make up 14% of treatment admissions.
Overall, then, addiction rates for marijuana are significantly lower than for other drugs, both legal and illegal. What about withdrawal symptoms? "You see mood effects, irritability. Food intake decreases. There are sleep disruptions. It looks like nicotine withdrawal," says Carl Hart, associate professor of clinical neuroscience at Columbia University, who has studied marijuana withdrawal. "You can actually die from alcohol withdrawal. Heroin withdrawal you can't really die from; it's more like the flu. Marijuana withdrawal is annoying, but it isn't life threatening." (Full disclosure: Hart and I are currently collaborating on a new book.)
According to Stanton Peele, author of the classic book Love and Addiction , the real question is not the substance's addictive quality, but its potential for harm. "I wrote an article on this titled 'Marijuana is addictive — so what?'" he says. "How harmful is this addiction compared to other addictions? It can be disruptive to people's lives; I have a treatment center, and some people end up there because of marijuana. On the other hand, in terms of physical assaults to your body, it's better than smoking and better than alcohol." ( More on Time.com: The Marijuana Number That Was Too Good to Check )
As Dr. Elders also said on CNN, marijuana is nontoxic. You can fatally overdose on alcohol, heroin or cocaine, but the only way a dose of marijuana will kill you is if someone crushes you under a bale of it.


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Burton, Richard, 1925-1984
From Library of Congress Name Authority File
Burton, Richard, 1925-1984
- [Richard Burton (1925-1984) was a Welsh actor.]
Instance Of
Scheme Membership(s)
Collection Membership(s)
- Jenkins, Richard, 1925-1984
- Jenkins, Richard Walter, Jr., 1925-1984
Additional Information
Birth Date
- (edtf) 1925-11-10
Death Date
- (edtf) 1984-08-05
Birth Place
Death Place
- male
Associated Language
- (itoamc) Actors
Exact Matching Concepts from Other Schemes
- found: Homage to Dylan Thomas. [Phonodisc] 1954.
- found: Burton, c1986: CIP galley (Richard Burton; d. 8/5/84)
- found: Internet movie data base, 30 Aug. 2002 (Burton, Richard; b. 10 Nov. 1925, Wales, named Richard Walter Jenkins, Jr.; d. 5 Aug. 1984; actor)
- found: IMDB, May 26, 2011 (b. in Pontrhydyfen, Wales; d. in Céligny, Geneva, Switzerland)
- found: The Official Richard Burton Website, Sept. 09, 2014 (Richard Burton was born Richard Walter Jenkins on 10 November 1925, etc.) {http://www.richardburton.com/life/}
- found: Wikipedia, Sept. 09, 2014 (Richard Burton, CBE; born 10 November 1925; died 5 August 1984; Welsh stage and cinema actor noted for his mellifluous baritone voice and his great acting talent. Establishing himself as a formidable Shakespearean actor in the 1950s, with a memorable performance of Hamlet in 1964; he was nominated seven times for an Academy Award without ever winning. He was a recipient of BAFTA, Golden Globe and Tony Awards for Best Actor. He remains closely associated in the public consciousness with his second wife, actress Elizabeth Taylor)
General Notes
[Richard Burton (1925-1984) was a Welsh actor.]
Change Notes
- 1980-08-12: new
- 2017-05-25: revised
Alternate Formats


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Diatoms are responsible for up to 40% of the carbon fixation in our oceans. The fixed carbon is moved through carbon metabolism towards the synthesis of organic molecules that are consumed through interlocking foodwebs, and this process is strongly impacted by the abiotic environment. However, it has become evident that diatoms can be used as 'platform' organisms for the production of high valuable bio-products such as lipids, pigments and carbohydrates where stress conditions can be used to direct carbon metabolism towards the commercial production of these compounds. In the first section of this review, some aspects of carbon metabolism in diatoms and how it is impacted by environmental factors are briefly described. The second section is focused on the biosynthesis of lipids and in particular omega-3 long-chain polyunsaturated fatty acids and how low temperature stress impacts on the production of these compounds. In a third section, we review the recent advances in bioengineering for lipid production. Finally, we discuss new perspectives for designing strains for the sustainable production of high-value lipids.
This article is part of the themed issue 'The peculiar carbon metabolism in diatoms'.
1. Diatoms and the use of carbon in their metabolism
(a) Availability of carbon in marine environment
Inorganic carbon (Ci) in the atmosphere has dramatically increased during the industrial era to exceed 400 ppm CO2 in 2016, whereas it did not exceed 300 ppm before the industrial revolution [1]. CO2 dissolution and biological carbon fixation (the so-called 'biological pump') make the oceans the main Ci sink on Earth. The former mechanism depends on atmospheric CO2 pressure, temperature, salinity and pH. At the usual pH of seawater, around 8.0–8.3, the dissolved inorganic carbon (DIC) is mainly in the form of , so that the equilibrium concentration of CO2 ranges between 10 and 20 µM [2,3]. Air-equilibrated seawater contains some 180 times more ionic Ci ( and ) than CO2 [4]. Altogether, the oceans have taken up approximately 30% of the anthropogenic CO2 emissions causing ocean pH decrease and temperature increase.
The biological pump relies on carbon fixation by photosynthetic organisms living freely or in symbiosis. However, CO2 concentration in seawater is low regarding the affinity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key enzyme of CO2 fixation. Indeed, the half-saturation constant of Rubisco measured in diatoms ranges between 30 and 40 µM [5]. To supply efficiently Rubisco, some marine diatoms, as well as other microalgae, can take up both CO2 and from seawater and concentrate CO2 close to Rubisco [6,7] using CO2-concentrating mechanisms (CCM) involving different carbonic anhydrase isoforms [8–12]. Moreover, a C4-like pathway highlighted in few diatom species could play a role in CCM [13–15]. Altogether, about half of the Earth's primary production takes place in unicellular microalgae [16]. Diatoms contribute up to 20% of global carbon fixation and thus play a major ecological role as primary producers [17–19]. When these organisms die, they settle on the seabed where carbon is sequestrated for geological timescales and might ultimately result in fossil fuels.
(b) Regulation of carbon metabolism: the influence of inorganic carbon availability
All the environmental factors such as temperature, irradiance levels and chemical compounds (heavy metals and nutrients) influence carbon metabolism. In this section, only the influence of inorganic carbon availability on diatom metabolism will be discussed. Regardless of the Ci form taken up by diatoms, Ci is finally reduced into carbohydrates during photosynthesis. These molecules are necessary for cell growth, respiration and synthesis of the other cell organic compounds (e.g. [20]). Diatoms export triose phosphates from the plastids and, under nutrient-replete conditions, store them in cytosolic vacuoles as the β-1,3-glucan polymer, namely chrysolaminarin [21,22] during the day, catabolizing this in the dark [23]. In diatoms living in microphytobenthic communities, carbohydrates largely contribute to the production of extracellular polymeric substances [24]. Changes of the environmental parameters that impact photosynthesis trigger modifications of the relative activity of the different metabolic pathways, resulting in lipid accumulation [25] that can increase the cell's buoyancy [22,26]. Caballero et al. [23] observed little change in chrysolaminarin accumulation or consumption during nitrogen starvation in Phaeodactylum tricornutum. Song et al. [27] demonstrated that gas flow rate in the culture medium plays an important role in P. tricornutum growth and production of lipid that is mostly composed of saturated and monounsaturated fatty acids (MUFA). When the / ratio increases to 0.125, the content of C16 : 1 increases to the highest value of 57%. The effects of CO2 concentration on C : N : P ratio in six diatoms show different trends according to the species and the light regime. In the range of dissolved CO2 from 1.5 to 37.7 µmol kg−1, either C : N and C : P ratios stay almost constant in P. tricornutum, decrease as in Thalassiosira weisflogii or increase in Amphora glacialis [28]. A CO2-dependent decrease in growth rate was observed at dissolved CO2 concentration below 10 µmol kg−1. Boelen et al. [29] reported that the photophysiological performance of the marine Antarctic diatom Chaetoceros brevis is not significantly affected by a pCO2 ranging from 190 to 750 ppmv under different irradiance regimes.
Although it is usually considered that diatoms are not mixotrophic [30], it has been reported that repeated additions of organic substrate such as urea [31] or acetate [32] can sustain mixotrophic growth of P. tricornutum UTEX-640 and Skeletonema costatum, respectively. Light intensity and quality impact on pigment contents, photosynthesis and nutritional value of diatoms [33,34], with photosynthesis/irradiance curves commonly used to characterize photoacclimation [35–37]. Generally, benthic diatoms have a lower maximum net photosynthesis but a higher light utilization coefficient than planktonic species [10,38]. Because chlorophyll a and carotenoid contents vary according to light environment and structural rearrangement inside chloroplasts [33,39], the motile benthic diatom Navicula cf. recens uses motility to select the optimal light exposure according to its photophysiological preferences.
2. Lipid accumulation and omega-3 long-chain polyunsaturated fatty acid biosynthesis in diatoms
(a) Glycerolipid biosynthesis
Many microalgae accumulate triacylglycerols (TAGs) in response to changes in environmental conditions. Depending on the cultivation conditions, some oleaginous species of diatoms have the ability to produce lipids up to 40–60% of the biomass and synthesize high content of highly valuable omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) such as eicosapentaenoic acid (20 : 5Δ5,8,11,14,17, EPA) and docosahexaenoic acid (22 : 6Δ4,7,10,13,16,19, DHA) (table 1). However, the mechanisms of the high lipid production in these organisms remain unclear. Fatty acid and glycerolipid biosynthesis have been studied in detail in the model plant Arabidopsis thaliana [51]. Similar pathways are predicted to occur in diatoms [52]. In photosynthetic organisms, the production of LC-PUFAs begins with de novo fatty acid synthesis in the plastids [53]. The first committed step is the formation of malonyl-CoA following the carboxylation of acetyl-CoA, catalysed by acetyl-CoA carboxylase (ACCase), a multi-subunit enzyme found in most prokaryotes and in the chloroplasts of plants and algae. The malonyl-CoA produced from the carboxylation of acetyl-CoA by ACCase is the central carbon donor for fatty acid synthesis. The fatty acid synthase (FAS) multi-subunit complex catalyses the production of C16 : 0 and C18 : 0 fatty acids (figure 1). Before entering the fatty acid synthesis cycle, the malonyl group of malonyl-CoA is transferred by malonyltransferase (MAT) to the cofactor acyl carrier protein (ACP) to form malonyl-ACP, which then undergoes a series of condensation reactions with acetyl-CoA, catalysed by a β-ketoacyl-ACP synthase (KAS) [51]. The candidate activities involved in fatty acid biosynthesis in diatoms have not yet been functionally characterized.
Each FAS cycle of reactions leads to the extension of the fatty acid chain by two carbon atoms, usually ending with the synthesis of saturated C16 : 0- and C18 : 0-ACPs. These acyl groups are then either retained in the plastids where they are transferred by chloroplastic acyltransferase to glycerol-3-phosphate (G3P) for incorporation into organellar membrane lipids (the so-called 'prokaryotic' pathway) or are hydrolysed from ACP by specific fatty acyl-ACP thioesterases (FATs) that release free fatty acids (FFA) in the inner envelope of the chloroplast. Some FATs can also release fatty acids containing upto 14 carbons. Alternatively, C16 : 0- and C18 : 0-ACPs can be desaturated by soluble palmitoyl- or/and stearoyl-ACP desaturases and subsequently released by FATs. These FFA are exported to the cytosol and are esterified to coenzyme A (CoA) by a long-chain acyl-CoA synthase (LACS) located in the outer envelope of the plastid to form acyl-CoAs [54]. The precise mechanism for this export step in algae is unknown and requires further investigation. By analogy with higher plants, it is predicted that these extraplastidic acyl-CoA esters are then transferred to endoplasmic reticulum (ER) or acylated onto a phospholipid backbone by the action of lysophosphatidylcholine acyltransferase (LPCAT) where they can undergo modifications before further participation in the synthesis of membrane lipids or storage TAGs ('eukaryotic' pathway).
In chloroplasts, the prokaryotic pathway generates PA and DAG and lipids such as galactolipids (monogalactosyldiacylglycerol, MGDG, and digalactosyldiacylglycerol, DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) [52,55]. Lipids synthesized in the chloroplast are characterized by the presence of a C16 acyl group at the sn-2 position of the glycerol backbone. In diatoms, the chloroplast is assumed to have originated from a secondary endosymbiosis event [52] and is surrounded by four membranes, representing a possible continuum between the ER and the outermost membrane of the plastid [56]. Therefore, the precise localization of the lipids synthesized via the prokaryotic pathway is difficult to predict.
In eukaryotes, TAG synthesis occurs via the Kennedy pathway [57] through the stepwise esterification of G3P and occurs in the microsomal fraction, presumptively the ER. The transfer of a fatty acid from the acyl-CoA pool to the sn-1 position of G3P is the first committed step in this pathway and is catalysed by glycerol-3-phosphate acyltransferase (GPAT), resulting in the production of lysophosphatidic acid (LPA). In plants and algae, the situation is further complicated by the presence of two types of GPAT, i.e. chloroplastic GPAT and extra-plastidial GPAT. Lysophosphatidic acid acyltransferase (LPAAT) catalyses the esterification of LPA at the sn-2 position of the glycerol backbone, giving rise to phosphatidic acid (PA).
Dephosphorylation at the sn-3 position of PA by phosphatidic acid phosphatase (PAP) generates diacylglycerol (DAG). Both PA and DAG are precursors for TAG and membrane phospholipids (mainly phosphatidylcholine, PC, and phosphatidylethanolamine, PE) biosynthesis. Diatoms also synthesize non-phosphorus glycerolipids, the so-called betaine lipids (BL), that are not found in higher plants [58]. BLs contain a polar amino-acyl group linked by an ether bond to the sn-3 position of the glycerol backbone. These lipids are presumably located in extra-plastidial membranes and may be involved in the transfer of fatty acids from the cytoplasm to the chloroplast. Comprehensive study of the glycerolipid content of P. tricornutum revealed the presence of diacylglyceryl hydroxymethyltrimethyl-β-alanine (DGTA), which has been found in some other algae [59]. Together with PC, it represents the major lipids present in the extra-plastidial membranes of P. tricornutum.
In plant seeds, large amounts of unsaturated fatty acids are synthesized on PC [60]. PC can undergo an acyl-editing cycle (the eponymous Lands cycle), by hydrolysis of the fatty acid at the sn-2 position, followed by a re-acylation with a fatty acid derived from the acyl-CoA pool [61]. Polar head exchanges can also occur by the action of a PC-DAG phosphocholine transferase (PDCT) [60]. In many plants, PC acyl editing and phosphocholine headgroup exchange between PC and DAGs control the majority of acyl fluxes through PC to provide PUFA for TAG synthesis [62]. It is unclear whether the same PC turnover occurs in diatoms, and given the abundance of BL, if that lipid also might act as either an acyl donor or receiver for this process.
The final step of the Kennedy pathway for the production of TAG is the acylation of 1-2-DAG on the sn-3 positions which can be achieved either with an acyl-CoA, catalysed by diacylglycerol acyltransferase (DGAT) or by transfer of an acyl-chain from PC by phospholipid : diacylglycerol acyltransferase (PDAT). Two types of DGAT isoforms, DGAT1 and DGAT2, are membrane-bound acyltransferases and play a major role in TAG biosynthesis. Most eukaryotic organisms contain a single copy of each DGAT gene, while photosynthetic microalgae appear to have multiple copies of DGAT2 genes: four putative DGAT2 genes have been identified in the diatom Fragilariopsis cylindrus and five isoforms have been found in P. tricornutum [63]. Accumulation of LC-PUFAs in glycerolipids has been studied in P. tricornutum [59] and Fistulifera solaris [64]. Complete characterization of P. tricornutum glycerolipidome in low-phosphate and low-nitrogen conditions demonstrated that the production of TAG most likely relied on a DAG substrate synthesized via the Kennedy pathway and on the combined activity of a DGAT and a PDAT adding a third acyl-group at position sn-3 [59]. A similar mechanism for EPA incorporation into TAG was observed in Fi. solaris, suggesting that EPA was desaturated on PC and the PC-based acyl-editing and headgroup exchange processes may contribute to the EPA incorporation into TAG [64].
Major activities of TAG biosynthesis have been annotated and partially characterized in several diatom genome sequences [65–67]. However, the precise subcellular localization of the diatom enzymes involved in fatty acid and glycerolipid biosynthesis continues to require further investigation.
(b) Biosynthesis of polyunsaturated fatty acids
The saturated fatty acids (C16 : 0 and C18 : 0) produced de novo can undergo further desaturation by plastidic or ER desaturases. Desaturation of C18 : 0 can be catalysed either by the soluble stearoyl-ACP Δ9-desaturase (SAD) of the chloroplast stroma [68] or by an extra-plastidic ER-bound acyl-CoA Δ9-desaturase (ADS) [69], generating oleic acid (OA, 18 : 1Δ9). OA becomes a substrate for further desaturation by Δ12-desaturase resulting in the production of linoleic acid (LA; 18 : 2Δ9,12) which subsequently may be converted into α-linolenic (ALA; 18 : 3Δ9,12,15) by the action of Δ15-desaturase (figure 2).
Linoleic acid and α-linolenic acid are essential fatty acids (EFA) and serve as the precursors for omega-6 and omega-3 LC-PUFAs, respectively [70]. The biosynthesis of LC-PUFA from LA and ALA may follow two converging routes: the predominant, or 'conventional' Δ6-pathway operating in most PUFA-synthesizing organisms, and an 'alternative' Δ8-pathway. To produce LC-PUFAs, a series of alternating desaturation and elongation steps are required. The 'conventional' pathway starts with Δ6-desaturation of both LA and ALA, resulting in the synthesis of γ-linolenic acid (GLA, 18 : 3 Δ6,9,12) and stearidonic acid (SDA, 18 : 4 Δ6,9,12,15), respectively. These two Δ6-desaturated fatty acids are then chain-elongated by two carbons via a Δ6-specific elongase which only accepts C18 fatty acids containing a Δ6 double bond, to yield di-homo γ-linolenic acid (DGLA; 20 : 3 Δ8,11,14) and eicosatetraenoic acid (ETA; 20 : 4 Δ8,11,14,17). A second desaturation, via the Δ5-desaturase, then produces arachidonic acid (ARA; 20 : 4 Δ5,8,11,14) and EPA.
Some marine organisms and protists use an 'alternative' Δ8-pathway in which LA and ALA are elongated by the action of a Δ9-specific elongase, yielding eicosadienoic acid (EDA; 20 : 2 Δ11,14) and eicosatrienoic acid (ETrA; 20 : 3 Δ11,14,17), prior to two sequential desaturations by a Δ8- and Δ5-desaturases to generate ARA and EPA, similar to the conventional pathway.
In DHA-accumulating microbes, the conversion of EPA to DHA may be carried out by the 'microbial' linear desaturation-elongation pathway [71]. In diatoms, EPA is most likely elongated by a specific Δ5-elongase to docosapentaenoic acid (DPA; 22 : 5 Δ7,10,13,16,19) which is then desaturated by a Δ4-specific desaturase to yield DHA.
All the corresponding genes encoding putative desaturases and elongases have been identified in diatoms and the presence of a highly active 'conventional' Δ6-pathway has been demonstrated for several diatom species such as P. tricornutum [72–75], T. pseudonana [76], F. cylindrus [77], Nitzchia sp. [78] and Fi. solaris [67]. Functional characterization of candidate activities involved in EPA and DHA biosynthesis from P. tricornutum and T. pseudonana indicates that these have the capacity to use both omega-6 and omega-3 intermediates to synthesize EPA and DHA [73,74,76].
LC-PUFA biosynthesis has been well studied in the model diatom P. tricornutum, in which the majority of EPA accumulates in polar lipids, especially in galactolipids such as MGDG and DGDG [59,79,80]. In vivo labelling in P. tricornutum revealed that EPA can apparently be synthesized by a number of different routes, involving Δ6- and Δ8-pathways with the predominant route proceeding via Δ6-desaturation of LA and using intermediates of ω6- and ω3-pathways [81]. Both Δ6- and Δ5-desaturation [73] and Δ6-elongation take place in the ER, and newly synthesized EPA is then imported into the plastids for incorporation into galactolipids [52,55]. Studies of the heterologous expression of P. tricornutum desaturases in yeast indicate that these are phospholipid-dependent activities (contrary to LC-PUFA elongases that use acyl-CoA as substrates), providing evidence that the substrate-dichotomy issue also exists in diatoms [72,82]. As the P. tricornutum lipidome contains only minor amounts of fatty acid intermediates of the EPA biosynthetic pathway, the accumulation of EPA as a single end-product is highly efficient in this diatom. Although P. tricornutum synthesizes small amounts of DHA and the putative gene encoding a Δ4-desaturase has been identified through sequence comparison, the activity of this candidate desaturase has yet to be confirmed. The absence of sequences associated with the 'alternative' Δ8-pathway may indicate that some of the activities associated with the conventional pathway also have (under some circumstances) the capacity to accept alternative substrates.
Similar sets of genes for LC-PUFA biosynthesis (i.e. Δ9-, Δ12-, Δ6-, Δ5-, ω3-Δ4 desaturases and Δ6-elongase) have been identified in the genomes of T. pseudonana [76], Fi. solaris (with the exception of Δ4 desaturase) [67] and in Nitzchia sp. transcriptome [78].
One unusual feature of the predicted pathways for LC-PUFA biosynthesis in two species of diatoms, P. tricornutum and Fi. solaris, is the apparent absence of predicted ω3-desaturases localized in the ER [55,83]. Such enzymes, including Δ15-, Δ17- and hypothetical Δ19-desaturases, convert ω6-fatty acids into their ω3-counterparts [84], and are considered to be essential for the enrichment of ω3-fatty acids over ω6 forms. In genome sequences for both these diatoms, only a single candidate gene for a plastidial ω3-desaturase has been identified. This raises the possibility of a dual localization of these putative plastidial ω3-desaturases, with access to both plastidial and ER-located substrates, similar to that found in Chlamydomonas reinhardtii [85].
(c) Regulation of eicosapentaenoic acid synthesis
During the past decade, all the primary activities required for the biosynthesis of LC-PUFA (elongases and desaturases) have been identified from multiple different species, and omega-3 biosynthesis has been successfully reconstituted in plants and microorganisms [86,87]. During PUFA synthesis, regulation occurs as a consequence of numerous factors [88] and environmental factors such as nutrients, light or temperature [89].
(i) Influence of carbon availability
A carbon source is absolutely required for lipid and fatty acid accumulation. Indeed, without this source, independently of nutrient deprivation, biomass or lipid synthesis is not possible. Diatoms, being photoautotrophic eukaryotes, use Ci such as CO2 as their carbon source and light as their energy source, thus participating in the reduction of atmospheric CO2 [20,90]. Bicarbonate has been shown to increase lipid accumulation in P. tricornutum [91]. This effect does not result from bicarbonate addition per se but from the concomitant pH increase [92]. Using 13C labelling, an equal incorporation of 13C carbon from bicarbonate has been observed in the C16 fatty acid chains incorporated into TAGs and PC, indicating that P. tricornutum is able to assimilate similar amounts of carbon regardless of nitrate availability. This suggests that an other internal lipid pool such as BL could be involved in TAG or PC synthesis [93].
In P. tricornutum, high levels of CO2 (0.15%) induce an increase in LC-PUFA levels, representing more than 16% of dry cell mass by comparison with around 7 and 8% obtained, respectively, with low (0.015%) and mid-CO2 (atmospheric, 0.035%) cultured cells [88]. The EPA content represents about 11.6% dry cell mass with high CO2 levels and 5–7% of dry cell mass under low and mid-CO2 conditions. However, the influence of CO2 concentration depends on the studied diatom species and specifically, the levels of EPA in the diatom concerned. Notably, P. tricornutum is rich in EPA and, consequently, levels of CO2 enhance EPA productivity. By contrast, Chaetoceros muelleri with low PUFA content [94] has no change in its EPA content when CO2 concentrations vary from 0.03 to 30% [95].
Mixotrophy with glycerol as a carbon source and ammonium as a nitrogen source enhances growth of P. tricornutum. The periodic supplementation of culture medium with glycerol and ammonium chloride increased the productivity of EPA by a factor of 10 when compared with photoautotrophically control conditions [96] (see also Finazzi [97]).
(ii) Influence of other nutrients
In response to salt stress, diatoms are able to change their lipid and fatty acid compositions to modify their membrane permeability. For example, Nitzschia laevis, Chen et al. [98] by testing different salt concentrations (from 10 to 30 g l−1) in Nitzschia laevis, have shown that the highest levels of EPA were obtained with a salt concentration of 20 g l−1, in parallel with the highest level of galactolipids and phospholipids, with EPA representing more than 70% of the total fatty acids in these polar fractions. These changes in the unsaturation of membrane lipids were suggested to be related to a modification of membrane permeability and fluidity under salt concentration as an acclimation to salinity stress.
Among nitrogen sources that can be used by diatoms, NaNO3, NH4Cl and urea have been tested in Cylindrotheca fusiformis and Cylindrotheca closterium. Whatever the nitrogen source, EPA levels were not modified, even though the maximum lipid accumulation was observed with NH4Cl in both species [99].
(iii) Environmental factors
Environmental factors have different effects on lipid and fatty acid production in P. tricornutum. Salinity, nitrogen concentration, light intensity or temperature can act differently on fatty acid composition and total fatty acid content. Indeed, with a salinity from 15 to 35 ppt or with different irradiance levels (50–150 µmol m−2 s−1), no changes in EPA levels have been observed after 7 days of growth. Decreased levels of EPA were observed under nitrogen limitation conditions (1.24 versus 12.35–49.40 mg l−1) or increased temperature (25°C versus 15°C) [100].
Light. In the marine diatom S. costatum, the highest amounts of EPA are obtained at the end of the logarithmic growth phase and during the stationary phase under high light levels (saturating irradiance) [32].
The quality and strength of UV irradiance also has distinct effects on fatty acid composition in diatoms. It has been reported that in benthic diatoms, even if modifications of fatty acid composition can occur after long-term (30 days) exposure to UV treatments, the UV-A exposure induces an increase in saturated fatty acids, while the UV-B exposure increased the unsaturated fatty acid (EPA) levels. Lipid content was increased after a short-term (6 h) UV-B exposure [101].
Temperature. Low temperature impacts lipid synthesis. For instance, when P. tricornutum is grown at 22°C for 8 days and then shifted to 10°C, the percentage of lipids increased [89] (table 2). Within 24 h after the temperature shift, a 61% increase was observed (table 2), suggesting that the mechanisms involved in the acclimation to low temperature are very rapid. The relative amount of EPA was modified in response to decreased temperature: it was increased by more than 17% within 48 h after the temperature shift and reached 38% after 4 days of thermal stress (table 2). The EPA enrichment can be even further increased by supplementing the growth medium with nitrogen and phosphate [89]. Similar results have been obtained with Odontella aurita. This diatom is used in aquaculture and in human nutrition [102]. In this taxon, the levels of DHA were also higher than when the diatoms were grown at room temperature [103].
3. Bioengineering in diatoms
Advances in next-generation sequencing technology and genetic transformation techniques together with transcriptomics, proteomics and metabolomics studies have greatly contributed to algal research. During the past few years, nuclear genomes of the pennate diatoms P. tricornutum, Fi. solaris and F. cylindrus and the centric diatom T. pseudonana have been sequenced, providing insights into the genome evolution of these distinctive groups [65–67,104]. Based on genomic and transcriptomic data, several metabolic networks for lipids have been constructed and PUFA biosynthetic pathways elucidated in the oleaginous diatoms P. tricornutum, Fi. solaris and Nitzschia sp. [55,56,67,78,83,105].
Recently, the first proteomic analysis of the oil-body-associated proteins in a diatom has been reported [106]. This study revealed several protein candidates associated with the oil bodies in oleaginous strain Fi. solaris and demonstrated that a proteomic approach may provide new insights into TAG accumulation in oil bodies.
Various genetic transformation systems have been developed in diatoms and several LC-PUFA synthesizing species have been successfully transformed by microparticle bombardment (Chaetoceros sp. [107], Cyclotella cryptica and Navicula saprophila [108], C. fusiformis [109,110], Fistulifera sp. [111], P. tricornutum [112,113], T. pseudonana [110]) and electroporation (P. tricornutum [114–116] and Chaetoceros gracilis [117]).
Several recent studies have demonstrated the advantages of a genetic engineering approach to increase the oil content and manipulate fatty acid composition in different species of diatoms (table 3). A number of strategies could be employed to regulate genes involved in lipid metabolism and LC-PUFAs biosynthesis.
During nuclear transformation, transgenes are integrated randomly, making targeting of specific genes more complicated. Rapid development of genome-editing techniques has greatly improved the potential for genetic engineering of microalgae. Targeted and stable modification of the P. tricornutum genome through disruption of UDP-glucose pyrophosphorylase has been achieved by using both meganucleases and transcription activator-like effector nucleases (TALENs) [132]. The mutant diatom produced more lipid (45-fold increase in TAG accumulation) than the control when grown in nutrient-stressed media. This generation of an enhanced lipid-producing strain demonstrates the power of genome engineering.
A similar successful example of using TALEN technology has been reported by Weyman et al. [133] who used TALENs to interrupt the gene encoding the urease enzyme in P. tricornutum. Metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. This technique allowed clarification of the role of urease in the urea cycle and also improved the molecular toolkit for diatom engineering. Other mechanisms for the targeted knockdown/out of endogenous genes include RNA silencing using RNAi and antisense RNA techniques [134], and high-throughput artificial-micro RNA (armiRNA) techniques [135].
Most recently, a highly efficient CRISPR/Cas9-based system has been developed to create stable targeted gene knockouts in P. tricornutum [136]. The reported genome editing method will allow cost-effective functional studies in microalgae and ultimately the engineering of specific traits.
Another valuable contribution to the development of novel tools for algal biotechnology is based on the introduction of an episome via conjugation into the cells of P. tricornutum and T. pseudonana [137]. A large DNA fragment has been delivered and maintained, suggesting that this new approach could facilitate metabolic engineering of entire biosynthetic pathways in diatoms.
(a) Overexpressing activities of fatty acid and triacylglycerol biosynthetic pathways
One of the first examples of direct targeting of lipid biosynthesis was the overexpression of an ACCase from the diatom Cyc. cryptica [127]. Despite a two to threefold increase in ACCase activity, no significant enhancement in lipid production in transgenic cells was observed. Based on these findings, it was considered that overexpression of ACC enzyme alone might not be sufficient to enhance the whole lipid biosynthesis pathway. One explanation that has been suggested is that under nitrogen stress, the build-up of precursors to the ACCases may play a more significant role in TAG synthesis than the actual ACCase levels [138].
Malic enzyme is involved in pyruvate metabolism and carbon fixation and should be taken into consideration when engineering microalgal strains with enhanced oil content. Overexpression of P. tricornutum malic enzyme (PtME) resulted in markedly increased lipid production in transgenic cells [135]. The total lipid content was enhanced 2.5-fold and reached 57.8% of dry cell mass with a growth rate similar to that of wild-type. The neutral lipid content was further increased by 31% under nitrogen-deprivation conditions. This study demonstrated a significant increase in the oil content in the transgenic diatom, suggesting a new target for developing microalgal strains for industrial production.
Another study demonstrated the important role of glycerol-3-phosphate dehydrogenase (GPDH) in lipid biosynthesis [136]. Overexpression of GPDH in P. tricornutum resulted in a 6.8-fold increase of the glycerol concentration per cell compared with the wild-type, indicating that the overexpression of GPDH promoted the conversion of dihydroxyacetone phosphate (DHAP) to G3P. There was a 60% increase in neutral lipid content, reaching 39.7% of dry cell mass in transgenic cells in the stationary phase, despite a 20% decrease in cell concentration. Fatty acid profiling showed that the levels of C16- and C18-MUFA significantly increased.
GPAT is the critical enzyme that catalyses the first step of TAG formation. A GPAT has been identified and functionally characterized in P. tricornutum [118]. Significant differences in lipid content and fatty acid composition were detected between transgenic and wild-type cells. The neutral lipid content determined by Nile red fluorescence staining was increased twofold. Fatty acid composition showed a significantly higher proportion of unsaturated fatty acids compared with wild-type. EPA levels increased by 40%, while those of C16 : 0 and MUFA decreased by 45% and 12%, respectively. These results suggest that the identified GPAT could efficiently upregulate TAG and biosynthesis in P. tricornutum.
Another approach included the direct modification of fatty acid composition through the expression of acyl-ACP thioesterases to control the chain length. Acyl-ACP thioesterases hydrolyse the acyl moiety of the fatty acid from acyl-ACP, releasing FFA. Heterologous expression of plant acyl-ACP thioesterases specific towards the generation of short-chain lauric acid (C12 : 0) and myristic acid (C14 : 0) in P. tricornutum resulted in an increased accumulation of shorter chain length fatty acids [125]. Similarly, overexpression of the acyl-ACP thioesterases with specificity for C18 fatty acids may improve LC-PUFA synthesis.
A putative thioesterase from P. tricornutum (PtTE; Phart2_33198) has been characterized and heterologously expressed in Escherichia coli, demonstrating moderate activity for C18 : 1-ACP and low activity for saturated fatty acids (C16 : 0 and C18 : 0) [126]. Overexpression of PtTE in P. tricornutum resulted in an almost twofold increase in both C16 : 0 and C16 : 1 and only a minor increase in C18 : 1 and EPA, enhancing the total fatty acid content to 72%.
Overexpression of acyltransferases could represent another valuable strategy for manipulation of lipid content and fatty acid composition in microalgae. An isoform of type 2 diacylglycerol acyltransferase (DGAT2), which participates in TAG assembly has been identified from diatom P. tricornutum [124]. Overexpression of DGAT2A in P. tricornutum resulted in the production of oil bodies, with a 35% increase of neutral lipids measured by Nile red fluorescence staining and a significant increase of EPA. The growth rate of transgenic microalgae remained similar, maintaining a high biomass. However, only one transgenic clone has been analysed and the role of DGAT2A in lipid enhancement requires further investigation.
Overexpression of biosynthetic enzymes of the LC-PUFA pathway may provide an effective way to improve the EPA and DHA content in marine microalgae. The first successful attempt in engineering the omega-3 trait in transgenic algae was reported recently on the overexpression of heterologous genes in P. tricornutum [120]. Phaeodactylum tricornutum can accumulate EPA to levels up to 35% of total fatty acid content, representing a good source for the industrial production of this valuable omega-3 LC-PUFA. However, P. tricornutum does not naturally accumulate significant levels of DHA. Heterologous expression of the Ostreococcus tauri C20 Δ5-elongase in P. tricornutum resulted in up to eightfold increase in DHA, and this level has been further increased with the co-expression of the acyl-CoA-dependent Δ6-desaturase, when compared with wild-type strains [120]. Importantly, DHA was shown to accumulate in TAGs, with several new TAG species detected in the transgenic strain. No detrimental effects to P. tricornutum growth rates were observed in transgenic cells: they were comparable to that of wild-type strains. This is the first report of enhancing the levels of omega-3 LC-PUFAs in a diatom by iterative metabolic engineering. This successful example indicates that metabolic engineering of diatoms represents a promising strategy for optimizing accumulation of beneficial omega-3 LC-PUFAs.
This transgenic strain of P. tricornutum (Pt_Elo5) has been further investigated for the scalable production of EPA and DHA [139]. Detailed studies were carried out on the effect of different media, carbon sources and illumination on omega-3 LC-PUFAs production by transgenic strain Pt_Elo5 grown in a 3.5 l bubble column, a 550 l closed photobioreactor and a 1500 l raceway pond with artificial illumination. In addition to ambient light, ponds were supplemented by halogen bulbs, light-emitting diode strips and fluorescent tubes (light level up to 110 µmol photons m−2 s−1) which were housed under polythene sheeting for culture/environment protection. A significant production of DHA over EPA has been observed in cells grown in a photobioreactor, equivalent to 2.1 mg l−1 DHA in a mid-exponentially growing algal culture. Omega-3 LC-PUFAs production in a raceway pond was an impressive 36% of TFA, averaging 23% EPA and 10% DHA (figure 3). These results clearly demonstrate the efficacy of large-scale cultivation of the transgenic Pt_Elo5 and is development as a platform for the industrial production of EPA and DHA.
When the Δ5-desaturase gene PtD5b was cloned and overexpressed in P. tricornutum, fatty acid composition was altered [123]. The neutral lipid content determined by Nile red fluorescence staining indicated an increase in transgenic cells. Engineered cells showed a similar growth rate to with the wild-type, thus keeping high biomass productivity.
A recent study has demonstrated that overexpression of endogenous fatty acid elongase genes in T. pseudonana can increase levels of EPA and DHA up to 1.4- and 4.5-fold, respectively [119].
(b) Blocking competing pathways
An alternative strategy to increase lipid accumulation is to block competing pathways to enhance the metabolic flux channelling to TAG biosynthesis.
Starch is a major carbon and energy storage compound in many microalgae. In diatoms, the fixed carbon is stored as the β-1,3-glucan, known as chrysolaminarin. This is a water-soluble polysaccharide stored in vacuoles. In diatoms, chrysolaminarin synthesis shares common carbon precursors with lipid synthesis. Thus, shunting carbon precursors from the chrysolaminarin synthesis pathway is an efficient way to boost oil accumulation [140]. UDP-glucose serves as the substrate for chrysolaminarin biosynthesis [141]. The effects of the suppression of UDP-glucose pyrophosphorylase (UGPase) on chrysolaminarin biosynthesis and carbon allocation in P. tricornutum were investigated in two studies. Daboussi et al. [132] reported a 45-fold increase in TAG accumulation in the transgenic strains, generated through the disruption of the UDP-glucose pyrophosphorylase using TALEN. A complementary study demonstrated that silencing of UDP-glucose pyrophosphorylase resulted in significant decreases in chrysolaminarin content and increases in lipid synthesis [128]. These findings suggest that UGPase is a rate-limiting enzyme and may play an important role in chrysolaminarin biosynthesis and carbon allocation. The results confirmed the feasibility of increasing lipid production through redirecting photosynthetically assimilated carbon away from starch synthesis to neutral lipid synthesis.
The pyruvate dehydrogenase complex (PDC) catalyses the conversion of pyruvate into acetyl-CoA, NADH and CO2, thus directing the carbon flow into the TCA cycle. PDC activity indicates the rate of the entry of pyruvate into the TCA cycle and subsequently to oxidative phosphorylation or fatty acid synthesis. PDC activity is primarily regulated by reversible phosphorylation by PDK, which phosphorylates and deactivates PtPDK [142]. Ma et al. [130] have identified a putative PDK gene (PtPDK) from P. tricornutum and generated a PtPDK antisense knockdown transgenic diatom. Neutral lipid content determined by Nile red staining of transgenic cells increased up to 82%, although the fatty acid composition was not altered. Interestingly, the transgenic cells showed slightly lower growth rate but similar to the wild-type cell size.
When starved for nutrients, microalgae redirect carbon towards biosynthesis of storage lipids, TAGs. Another strategy to develop an efficient pathway to enhance lipid production is by suppressing a diatom's ability to incorporate nitrogen. Levitan et al. [129] have studied the effect of nitrogen stress on the remodelling process in P. tricornutum by knocking down the gene encoding for nitrate reductase, a key enzyme required for the assimilation of nitrate. The transgenic strains exhibited 40–50% of the mRNA, protein content and enzymatic activity of the wild-type, concomitant with a 43% increase in cellular lipid content. This study indicated that a nitrate reductase knocked-down strain shunted approximately 40% more carbon towards TAGs than the wild-type without losing photosynthetic capacity, suggesting that redirecting carbon towards lipid biosynthesis can be achieved by altering the expression of genes distinct from those directly involved with lipid biosynthesis.
An alternative strategy to increase lipid accumulation is to decrease lipid catabolism. The targeted knockdown of lipid catabolism, and specifically lipases, could potentially improve the accumulation of FFA and enhance lipid content with less impact on the primary carbon pathways associated with growth. Trentacoste et al. [131] using antisense RNAi constructs successfully engineered T. pseudonana to disrupt lipid catabolism by targeting an enzyme with lipase activity. This targeted knockdown resulted in an increase in the accumulation of TAG and total lipid yield without impacting growth rate, under both continuous light and alternating light/dark, continuous growth and nutrient replete versus nutrient deficient conditions. Fatty acid analysis revealed that knockdown strains contained more 16 : 0 and 18 : 2 than wild-type species. However, the mechanisms responsible for this skewing of the fatty acid profile are unclear. These results demonstrate that disrupting lipid catabolism is also a feasible approach to improve TAG accumulation in microalgae without affecting growth rates or biomass production.
Most microalgae are obligate photoautotrophs and require light for their growth. The requirement for light to increase cell densities and production is the main objective and a limiting factor of their cultivation [48]. Heterotrophic growth in conventional fermenters can be a cost-effective alternative to photoautotrophic growth. Zaslavskaia et al. [143] performed a trophic conversion of P. tricornutum by introducing a gene encoding a glucose transporter and demonstrated that transgenic P. tricornutum could thrive on exogenous glucose without light, thereby becoming heterotrophic.
Recently, a similar approach has been employed to create a heterotrophic strain of P. tricornutum that also accumulates enhanced levels of DHA [144]. This was achieved by generating transgenic strains co-expressing Δ5-elongase from O. tauri with a glucose transporter from the moss Physcomitrella patens. This double transformant has the capacity to grow in the dark in liquid medium supplemented with glucose and accumulate substantial levels of omega-3 LC-PUFAs. The effects of glucose concentrations on growth and LC-PUFA production of wild-type and transformed strains cultivated in the light and dark were studied. The highest accumulation of omega-3 LC-PUFAs was observed in cultures grown under mixotrophic conditions in the presence of 1% glucose (up to 32.2% of total fatty acids). Both DHA and EPA were detected at high levels in the neutral lipids of transgenic cells grown under phototrophic conditions, averaging 36.5% and 23.6% of total fatty acids, respectively. This study demonstrates the potential for P. tricornutum to be developed as a viable commercial strain for both EPA and DHA production under mixo- and heterotrophic conditions.
4. Conclusion
During the past decade and a half, intense research has been performed and considerable progress made in the genetic engineering of diatoms. Based on recent advances, it is clear that rational metabolic engineering of microalgae has a promising future for designing strains with improved lipid accumulation and desired fatty acid content. It is likely that a combination of strategies will provide the most feasible approach to improve LC-PUFA production and accumulation in TAG without inhibiting growth rates. In particular, our ever-improving understanding of lipid metabolism, combined with the promise of synthetic biology approaches (design–build–test), increased automation and HTP analyses, have the capacity to provide a literally transformative shift in our ability to direct the biosynthesis of any chosen compound. Ultimately, a digital blueprint of diatom metabolism would be available which would be predictive for varied inputs and outputs in (e.g.) lipid biosynthesis. By such computationally supported approaches, the true promise of predictive biology to deliver useful outputs will be realized.
Data accessibility
This article has no additional data.
Competing interests
We declare we have no competing interests.
This project was supported by BBSRC grant no. BB/L002957/1.
Rothamsted Research receives grant aided support from the Biotechnology and Biological Sciences Research Council (BBSRC), UK.
One contribution of 16 to a theme issue 'The peculiar carbon metabolism in diatoms'.
Electronic supplementary material is available online at https://dx.doi.org/10.6084/m9.figshare.c.3800209.
- Accepted April 14, 2017.
- © 2017 The Authors.
Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.


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Squirrel's Fall SearchBook - 2012
Scamper in the colourful fall leaves with Squirrel. Squirrel loves fall. He chases swirling leaves and collects food with his little brother. Squirrel has so much fun that he forgets where he puts his food! Will he find it again before his little brother eats it all? Series Description: Beautifully presented series of picture books, designed to entertain as well as to inform young readers about the essence of each of the four seasons. Suitable for reading together as well as reading alone, these books are not only highly enjoyable picture storybooks but also a learning tool for literacy and the natural world. Animal Season series includes: Mouse's Summer Muddle 978-1-60992-226-9, Rabbit's Spring Adventure 978-1-60992-225-2, Squirrel's Fall Search 978-1-60992-227-6, and Owl's Winter Rescue 978-1-60992-224-5
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Hurricane Ridge Ski Racing & CompetitionsPort Angeles, WA US
Contact Info / Directions
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Ski Race Programs at Hurricane RidgeHurricane Ridge has a home and traveling Alpine Ski racing team, participating in local and regional races, regularly sending athletes to championship races.
Races and Competitions at Hurricane RidgeNo upcoming events posted.
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Be the first MLive.com user to add photos or videos of Walke Lacey MD PC
Walke Lacey MD PC is located at 28111 Hoover Rd Ste 1A, Warren, MI. This location is in the Northeast Warren neighborhood. This business specializes in General Practice Medicine.
Walke Lacey MD PC can be found at Hoover Rd 28111. The following is offered: General Practice Medicine. The entry is present with us since Sep 9, 2010 and was last updated on Nov 14, 2013. In Warren there are 44 other General Practice Medicine. An overview can be found here.


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Graphics Collection and Photographic Archives
50,000 items, 16th —21st century
The Graphics Collection and the Photographic Archives comprise about 50,000 watercolours, drawings, graphic prints and photographs, including a collection of national importance of about 150 daguerreotypes (photographs on copper), ambrotypes (photographs on glass) and pannotypes (photographs on waxed cloth).
The main focuses of the Graphics Collection and Photographic Archives are the historical topography of the City of Berne, its surroundings, Bernese country estates and Bernese portraits. Themes from arts and crafts, cultural history and other areas are also represented. The biggest individual collections are the postcards and over 11,000 negatives from the photographic archives of the postcard publisher Franco-Suisse.
The Burgerbibliothek of Berne also holds private archives and partial bequests from artists, including Friedrich Walthard (1818—1870), Rudolf Münger (1862—1929), Ernst Kreidolf (1863—956), Victor Surbek (1885—1975), Paul Boesch (1889—1969), Rudolf Mumprecht (*1918) and Daniel de Quervain (*1937).
Please book in advance to use the collection.
Topographic content and subdivision by subject
City of Berne
The collection comprises:
- Topographic views from the 16th century onwards, predominantly historic town plans from the 19th and 20th centuries
- Architectural drawings, including those by Erasmus Ritter (1726—1805) and Albrecht von Stürler (1705—1748)
- Photographs, including by Friedrich Gerber (1797—1872), Carl Durheim (1810—1890), Hermann Völlger (1855—1930) and Moritz (working 1860—1894) and Emil Vollenweider (1849—1921)
Canton Berne and the rest of Switzerland
The collection comprises:
- Views of patrician estates in the canton of Berne
- Cultural-historic images and photographs (regional traditions, costumes, farmhouses) of canton Berne
- Topographic views from the rest of Switzerland
- Maps and plans (national maps, tithe maps, forest maps, building plans)
- Photographic collections including by Albert Stumpf (1867—1951) and Jean Moeglé (1853—1938)
The views from abroad are mainly associated with the following names:
- Theodor Zeerleder (1820—1868), drawings and watercolours
- Horace Edouard Davinet (1839—1922), collection of stereo photographs
- Cécile (1855—1929) and Eduard (1849—1926) von Rodt, photographic collection
Subject areas
All the images are indexed by subject area, even where they mainly depict topographic features. Subject areas include: occupations and manual work, folk art including crafts and customs, coats of arms and genealogy including family trees, Gotthelf illustrations, plants and animals and many more.


text: After losing all my fonts (don't ask) i'm rebuilding my entire library from scratch and although i've bookmarked various links to cool fonts i've seen in the past i was wondering if anyone had any recommendations as to collections of fonts they can't live with out. For example there's the obviously ubiquitous Adobe Font Folio but does anyone know of any smaller (cheaper) collections available of solid usable fonts they've come to love? I'm especially interested in usable geometric collections such as: http://www.smashingmagazine.com/2007/11/08/40-excellent-freefonts-for-professional-design/ All recommendations are much appreciated


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Ben of Xboxer360 writes, "Since receiving the mysterious invite, emblazoned with the notorious Assassin's logo, to what could only be an Assassin's Creed event, I was excited. Once there, I was ushered into a small smoke-filled room, tinted blood-red and decked out with the armour of great Assassin's from our past. Altair, Ezio, Connor, the room steeped in Assassin's history.
My anticipation was building and after a short but heart-felt monologue from a great British thespian, the room heard a bang before the walls were covered in falling black pirate flags. These weren't just any pirate flags, the crisp Assassin's logo was etched onto the skull and cross-bones.
This is Assassin's Creed 4: Black Flag"
I understand why people are annoyed at this game coming so soon but I can't help but get more excited the more I hear. Being a pirate is going to be awesome.


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Hwy 10 eastbound lanes closed between Detroit Lakes and Audubon
BEMIDJI, Minn. – (8:00 p.m.) The Minnesota Department of Transportation has closed the eastbound lanes of Highway 10 between Detroit Lakes and Audubon.
The lanes are anticipated to be closed until tomorrow morning and motorists are advised to seek alternate routes. The detour follows County Road 15, County Road 6 and Highway 59.
For real-time travel information and road conditions anywhere in Minnesota, visit www.511mn.org.


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RELEASES, ETC.: Before being declared a winner, a selected entrant will be required to sign a Declaration of Compliance and a Release of Liability form, which (among other things): (i) confirms compliance with these Official Contest Rules; (ii) acknowledges acceptance of the applicable Prize as awarded; (iii) releases the Sponsor, its advertising and promotional agencies, any contest judging organization, parent companies and affiliates of the foregoing and all of their respective directors, officers, owners, partners, employees, agents, representatives, successors and assigns (collectively, the "Releasees") from any and all liability in connection with this Contest, his/her participation therein and/or the awarding, use and/or misuse of the Prize or any portion thereof; and (iv) agrees to the publication, reproduction and/or other use of his/her name, address, voice, statements about the Contest and/or photograph or other likeness without further notice or compensation, in any publicity or advertisement carried out by or on behalf of the Sponsor in any manner whatsoever, including print, broadcast or the internet. The Releasees do not make, nor in any manner are responsible or liable for: (i) any warranty, guarantee or representation, expressed or implied, in fact or in law relative to any Prize; and (ii) are not liable for injury, loss or damage of any kind resulting from the acceptance, use and/or misuse of any Prize, travel related thereto (as applicable), or otherwise from participation in this Contest. Declaration and Release documents must be returned within the time period indicated in the documents or the applicable Prize will be forfeited.
This contest is in no way sponsored, endorsed or administered by, or affiliated with Apple, the App Store, iOS, Google, the Google Play store, or Android.
LIMITATIONS OF LIABILITY: Without limiting the limitations of liability set forth elsewhere in these Official Contest Rules and/or the Declaration of Compliance and Release of Liability, and for greater certainty, the Releasees will not be liable for: a) any incomplete or inaccurate information, whether caused by Contest Website users or by any equipment or programming associated with or utilized in the Contest, or by any technical or human error which may occur in the capturing and/or processing of Entries; b) the theft, loss, destruction or unauthorized access to, or alteration of, Entries; c) any problems with, or technical malfunctions of, telephone networks or lines, computer on-line systems, servers or providers, computer equipment, software, viruses or bugs; d) any failure of any e-mail to be received by or from Sponsor and/or Entry to be captured or recorded for any reason including but not limited to traffic congestion on the Internet or at any website or combination thereof; e) any typographical or other errors in the offer or administration of this Contest, including but not limited to errors in advertising, these Official Contest Rules, the selection and/or announcement of eligible winner(s), and/or the distribution of any Prize(s); and/or g) any combination of the above.
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INTELLECTUAL PROPERTY: All intellectual property, including but not limited to trademarks, trade names, logos, designs, promotional materials, web pages, source code, drawings, illustrations, slogans and representations are owned by the relevant Sponsor and/or its affiliates. All rights are reserved. Unauthorised copying or use of any copyrighted material or intellectual property without the express written consent of its owner is strictly prohibited.
COPYRIGHT-PROTECTED WORK: By submitting an Entry into the Contest, the entrant represents and warrants that the Entry submitted is original to the entrant, and all right, title, and interest (including copyright) thereto is owned and/or controlled by the entrant to the full extent necessary to enable the Sponsor to use the Entry as contemplated herein; and that the Entry does not infringe upon the intellectual property or other statutory or common law rights of any third party. In consideration for the opportunity to participate in the Contest, the entrant hereby (i) grants to the Sponsor, in perpetuity, a non-exclusive license to publish, display, reproduce, modify, edit or otherwise use his/her Entry, in whole or in part, for advertising or promoting the Contest, or, for any other reason; (ii) waives all moral rights in and to his/her Entry in favour of the Sponsor (and anyone authorized by the Sponsor to use the Entry); and (iii) agrees to release, indemnify and hold harmless the Releasees from and against any and all claims related, directly or indirectly, to his/her Entry – including, without limitation, claims based on publicity rights, defamation, invasion of privacy, copyright infringement, trade-mark infringement or any other intellectual property related or other cause of action.
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Lazy Apple Pie
- Prep: 5 min
- Cook Time: 60 min
- Total: 1 hr 5 min
- Serving: 8 servings
Lazy Apple Pie is for the working woman who wants a pie, wants it now and wants it fresh and delicious. Yes, there are shortcuts. No, taste is not sacrificed.
- 2 High Quality Frozen Deep Dish Pie Shells (I prefer Marie Callendar)
- I can prepared apple pie filling
- Cinnamon Sugar
- Preheat oven to 325 degrees
- Thaw pie shells
- Pour apple pie filling in pie shell and sprinkle with cinnamon sugar
- Carefully remove remaining pie shell from container. (This is the hard part) and place it on top of the apple-filled shell.
- Pinch sides of crush together to seal. By the way, if you happened to tear the shell as you were removing it from the pan, just pinch it together. It will be fine.
- Poke a few holes in the top of the pie and sprinkle with some more cinnamon sugar
- Bake for about an hour.
Filed Under:
Dessert Recipes, Thanksgiving Recipes


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Just as health departments across the nation are warning that this year's milder winter bring an especially high threat from ticks in the spring and summer, federal health officials say two new cases of a tick-borne illness that kills 1 in 10 infected have been reported in Maine. WND reported last month the Centers for Disease Control warned of the emergence of a far deadlier tick-related virus than Lyme Disease that kills 10 percent of those infected and permanently
disables another 50 percent. It's called POW for short, or Powassan, and it, like Lyme, is carried by deer. According to a Fox News report, the cases in Maine bring that state's count of infections to nine since 2000. The patients were not identified by the CDC, but officials said the concerns are legitimate because the virus differs from Lyme disease in that it can be transmitted through a tick bite in only minutes. READ MORE


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Dynegy Inc. said Tuesday that one of its subsidiaries is seeking bankruptcy protection as part of a plan that will force bondholders to take heavy losses while shielding major investors including billionaire Carl Icahn.
The Houston power company, which serves utilities in the Midwest, Northeast and the West, made the decision after struggling for years with lower electricity prices, falling credit ratings, and infighting among investors over competing takeover offers.
As part of its bankruptcy filing, Dynegy said it has agreed with some of its bondholders on a plan that would restructure $4 billion in debt. The deal "will significantly reduce the amount of debt on the company's consolidated balance sheet," Dynegy said in a statement.
It allows the company to deal with its debts while keeping its coal and gas-fired power plants running in "a fashion that will maximize our operational flexibility," Dynegy President and CEO Robert Flexon said in a statement.
None of its power generating businesses will be impacted by the bankruptcy filing, the company said.
As debts rose to more than $5 billion this year, Dynegy worked behind the scenes to protect the company. Instead of selling assets to raise cash, the company reshuffled its businesses so that the parent company owned power plants and other assets while a subsidiary issued the bonds.
Bondholders have cried foul, and in September, they filed a lawsuit in the Supreme Court of the state of New York alleging that Dynegy illegally protected some assets from claims they could make in bankruptcy court.
Meanwhile, the company tried to negotiate with bondholders, offering them a deal that analysts said would have forced bondholders to lose 60 percent to 72 percent of what they were owed. That deal fell apart last week, and the company decided not to make a $43.8 million interest payment.
Dynegy said Tuesday that Dynegy Holdings LLC and four of its subsidiaries have filed a voluntary petition in the U.S. Bankruptcy Court for the Southern District of New York. The parent company and its other subsidiaries are not seeking relief, and Dynegy said they will continue to operate with no impact from the limited Chapter 11 filings.
Dynegy said the agreement focuses on a framework for restructuring obligations owed by Dynegy Holdings. Subsidiaries of Dynegy Holdings involved in the filing include Dynegy Northeast Generation Inc., Hudson Power LLC, Dynegy Danskammer LLC and Dynegy Roseton LLC.
Dynegy sells electricity to grids and utilities in the Midwest, the Northeast and the West.
Shares increased 50 cents, or 17 percent, to $3.45 in premarket trading. They tumbled 11 percent Monday in anticipation of the bankruptcy announcement.


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LightningDiamond wrote: I've been looking for sunstone and only know of a few bosses that drop them: The Trial of Spheres Bosses and the Pylon Bosses in Stormriven. Other than that i dont know of any. I've heard some people say they found them off plants but i've yet to see someone list the specific plant. So if anyone knows of the plant or other ways of obtaining sunstone please share! :-(
Happyyoshi wrote:I myself have needed sunstone for my last grandmaster artisan badge but I did manage to do a pretty easy plan I went bought sandstone if they had it at the bazaar and if i needed more but there was non I went to look for stoneblocks and or buy stoneblocks once again from the bazaar and then transmute them into sunstone now I just need some golden pearls hahahaha hope this helps.~Chris?IceBlade
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City of Eden PrairieHome Menu
Community Center Room Rentals
The City of Eden Prairie offers rental facilities available for every occasion, from intimate meetings to special gatherings.
The Community Center offers spaces available for rent that can accommodate groups from 10 to 100 with premier venue areas including the Cambria Room and Gymnasium. Spaces are available to rent on an hourly basis with a 2-hour minimum reservation. The Community Center offers wireless Internet throughout the building. The Cambria Room, Room 201 and the 101 Boardroom have built-in A/V systems. A portable A/V system is available upon request for use in the other rooms. Amenities vary per room, click the name of the room below for more information.
Community Center Room Rentals FAQ
Approved Caterers [PDF]
Guidelines for Community use of Parks and Recreation Rental Facilities [PDF]
Room 202
- Address:16700 Valley View Road
Eden Prairie, MN 55346
- Phone:952-949-8333
- Wireless Internet
- Community Center Room Rentals
- Address:16700 Valley View Road
Click here to check availability
Click here to view additional photos
Room 202 can accommodate 25 auditorium-style, 15 classroom-style, or 12-15 in a boardroom or horseshoe arrangement. Projector available upon request.
Fees (fees are per hour and require a 2-hour minimum, tax not included)
Eden Prairie Non-Profit: $25
Eden Prairie Business or Resident: $30
Non-Resident or Non-Eden Prairie Based Group: $45
Still Need to Pay for Your Reservation?
If you've made a reservation but still need to submit payment online, please visit edenprairie.org/Register, login to your account and click the shopping cart icon to proceed to checkout with your Visa, MasterCard or Discover card. If you've misplaced your username and/or password, click the "Contact Us" button at the top of this page to request your login information.


text: You see it all over the web. People upset about how much they pay for Gatorade. I always thought, "Then why do people put up with it?" If it's just water, sugar and salt, make it yourself!" Well, got to give credit to this guy at this blog called Cheap Eats. He actually tried to make homemade Gatorade -- at a cost of 18 cents. Turns out the experiment didn't really work out as his Gatorade tasted like, in his own words, Orange Flavored Ocean. Check out the complete entry here: https://www.bloglander.com/cheapeats/2006/08/01/homemade-gatorade-recipe


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Here is the description about various Episodes in View Source.
Andy Boyle, who is a web developer of newsroom explains how code was cobbled together by him to create a application of people locator in the wake of the Tuscaloosa tornados which was occurred in 2011.
David Cohn who has helped several weeks ago to coordinate an occasion named TechRaking. A lot of dialogues called for the belief that journalists are distinct because journalists do not have a culture of neglecting freely to enhance that day. This podcast is dedicated to that purpose and perform those things, speaking regarding the failure of digital journalism. Successes will also be discussed, however there isn't any amazing job that does not have a boatload of neglect in the day's aftermath.
- Django Podcast Show and Case sensitive episodes are other Podcasts by 3rdaverad.io.
At the Chicago Tribune, the designer of news application is none other than Heather Billings. He speaks considering replumbing WordPress to function the need for self learning as well as more like Django.
Custom e-mail feeds as well as the newsletters can be created by The Spokesman Review. It might be sounded not very well, but there are enormous consequences for reader-managed news.
- Love Music? Then find Music Venues near you in United States.
Ben Welsh of the la Times takes you throughout the data gathering, evaluation and generation procedure to produce the "How quick is LAFD where you live?" map.
The Los Angeles Times' Ben Welsh will drive you through the collection of data, production and analysis process to create the map of "How fast is LAFD where you live?".
Homicide Watch's Chris Amico will take you throughout the Your Warming World Project creation for NewScientist.


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All You Need to Know About Shoelaces
You probably don't give shoelaces much thought because they are slowly disappearing from sight. They are being replaced by Velcro, which will eventually be replaced by spray on shoes. In the long run, the lowly shoelace will be tied to other obsolete antiquities such as the buggy whip.
Yes, there will be shoelace collectors and a few colorful pairs behind glass in museums. But you will not find them on shoes.
Then, someday in the distant future, a college student will need to write a paper about the "ancient" shoelace of the 21st century. He or she can then use this article as a shoelace reference source.
So gather round as I string together the tale of those quaint pieces of fabric that used to hold shoes onto people's feet.
A Brief History Of The Shoelace
Unlike the chicken and egg question, shoes came before shoelaces. "Shoes" originally were just a piece of some animal's hide cut to fit on the bottom of peoples' feet.
Caveman Urg: "What me do, Gurg? Feet hurt when I step on rock."
Caveman Gurg: "Hide."
Urg: "Why hide? You see saber-tooth tiger?"
Gurg: "No, put hide on feet, you Neaderthal!"
But Urg did not know how to keep the hide from falling off his feet. So Gurg probably came up with a long piece of dried animal guts or hide, or a long strip of grass or bark to hold the hide in place. And so the shoelace was invented.
As shoes became more sophisticated, dried animal gut would no longer do. Shoes were secured with buckles, buttons, and ribbons. By the way, a buttonhook was used to close shoes that used buttons. Alas, the shoe buttonhook is long gone, much as the shoelace will be in the future.
Then, in 1790, a man named Harvey Kennedy received a patent for a shoelace. And so began the modern era of the shoelace.
Shoelace Bits and Pieces
The first materials for shoelaces were the same stuff used to make rope: hemp, cotton, and leather. Cotton and leather shoelaces are still around. In fact, I use leather laces on my topsiders.
But most shoelaces are now made with synthetic fibers. Synthetics are used because they can last longer and are less prone to rot than the other kinds of shoelace materials. Another big plus is that the synthetic fibers make shoelaces more slippery that the other materials.
Why do you want slippery laces? You can tie them faster. On the other hand, do you wonder why your laces keep coming undone? Blame it on the slippery material.
On the shoe end of things, people needed a way to get the shoelaces to stay on their shoes. The solution was eyelets, through which you guide the shoelaces. Originally, eyelets were sewn holes using thread. Then, in the mid-1800s, shoe manufacturers began using metal eyelets because they could all be the same size and took less labor to put on the shoe.
At the tip of shoelaces, you'll find small plastic or metal tubes. These are called aglets. Their purpose is to help those shoelaces slip more easily through those eyelets on the shoes.
So you've got your fabric laces, eyelets, and aglets. Quite a neat little system, eh?
Shoelace Lengths
Did you ever wonder why shoelaces come in different lengths? Probably not, but here's the answer anyway. Shoe designers make shoes in different styles and for different purposes, such as fashion, walking, hiking, and running. Different styles and types of shoes require different numbers of eyelets, which, in turn, require different lengths of shoelaces.
So, for you researchers from the future, here is a handy-dandy chart that tells you what length shoelace you should use depending on the number of eyelets on your shoes.
Number of pairs of eyelets
Shoelace size - dress shoe
Shoelace size - sneaker
24" or 60 cm
28" or 70 cm
28" or 70 cm
32" or 80 cm
28" or 70 cm
35" or 90 cm
32" or 80 cm
39" or 100 cm
32" or 80 cm
43" or 110 cm
35" or 90 cm
47" or 120 cm
47" or 120 cm
51" or 130 cm
Lacing Methods
When teaching kids how to tie shoelaces, parents keep it pretty simple. (Read this Hub about tying shoelaces for more info.)
But adults may want to get fancier when tying their own laces.
Caveman Urg: What you got on feet hide?
Caveman Gurg: Got gut shoelaces tied fancy style.
Urg: Why you need fancy style laces?
Gurg: Need hide look good when clubbing.
The moral of the story is that different activities and tastes require different lacing styles.
You probably know the following lacing methods:
- Straight
- Criss cross
- Over under
- Army style
But there are lots of other more exotic lacing methods such as the:
- Starburst
- Asterisk
- Roman
If you want to learn how do to these and other exotic lacing methods visit this site.
Other Uses for Shoelaces
You would think that with a name like "shoelace" people would only use them for tying shoes. But no! There are many other uses for shoelaces.
People have used shoelaces:
- For committing suicide (Why do you think they're not allowed in prison cells?)
- As a belt
- As a bracelet
- For a hairband
- For tying sneakers together so you can throw them over telephone wires
In fact, there are lots of arts and crafts projects you can do with shoelaces. Visit this site to see a few more.
Goodbye Shoelaces
And so, as Velcro secures the end of shoelaces, let us raise our glasses and tie one on for the lowly shoelace.
Magic Trick with Shoelaces
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BILSTEIN B14 - PSS Coilovers for BMW MK5 (E39)
Product Code: 47-111264
Availability: In Stock
535 i, 540 i (04/96 - 06/03)
Sport makes you flexible. With the BILSTEIN B14 threaded ride height adjustable kit, we?re bringing technology from the race track onto the street.
Threaded body adjustable range of 20 mm at both axles, while installed Lowering of approx. 30-50 mm at front and rear Road-tested by BILSTEIN under motor sports conditions and fine-tuned Spring plates and lock nuts made from a special aluminium alloy Surface coating using Triple-C-Technology© for long-lasting corrosion resistance Round threads for ease of adjustment Quality sport springs made from highly durable material BILSTEIN gas pressure technology BILSTEIN mono-tube/Upside-Down technology, made in Germany German TV certificate
without ride height adjustment; with standard chassis; with M-technics; without electronic suspension control; Spacers required for several wheel/tyre combinations Front lowering: 25-50 mm; axle load to: 1105 kg Rear
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2016 Conference Sponsors
We are looking for conference sponsors – agencies, organizations, foundations and companies – that are leading the way in creating, developing and implementing communities and neighborhoods that are both pike and pedestrian friendly, and are safe environments for our children to live, plan, and go to school. Conference opportunities offer many ways to interact with participants and underscore your commitment to the important issues to be addressed through this event.
Download the sponsorship brochure for more information.
For more details please contact Amanda McEldowney at email@example.com.
Thank you to our Sponsors!
Program Sponsor:
Network Sponsor:
Supporting Sponsor:
Workshop Sponsors:
Celebration Sponsors:
www.tooledesign.com www.murphyepson.com
www.kleingers.com www.transystems.com
Partner Sponsors:
www.murphyepson.com www.kleingers.com www.nationwidechildrens.org
www.transystems.com www.mbakerintl.com www.msconsultants.com
www.franklincountyohio.gov www.dero.com
www.drivingskillsforlife.com www.tooledesign.com


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Title: Memorial to John Brown at Quindaro Townsite, Kansas City, Kansas
Alternate title: John Brown Memorial
Description: Lifesize marble statue of John Brown, which was dedicated on June, 1911, at Western University campus in Quindaro Townsite, Kansas City, Kansas.
Date original: 1911
Place: Quindaro, Kansas
Person: John Brown
Owning organization: Wikimedia Commons
Document Type: image
Publication Statement: http://commons.wikimedia.org/wiki/File:BrownMemorial1911.png
Organization location: Photograph courtesy of J.D. Redding. Image licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license. This license grants permission to copy, distribute, transmit, and adapt the work. Use of this image requires attribution in the manner specified by the author or licensor. Alteration, transformation, or adaptation of this work may be distributed only under the same or similar licence of the original.
Page Type: Digital Repository ObjectShow Extra Info
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March 15, 2010 | Volume 5 Issue 5
• TOP STORY: Ease of Use Takes on New Meaning with Visual LANSA V12• FEATURED ARTICLE: Technology Focus: Brave, Courageous, and Bold: Qualities You May Need to Select an ERP Application• MORE NEWS: IBM Achieves Highest Growth Among Top Three Storage Vendors • FORUM: Great News for Users of System i XLPARSE • EVENT: WMCPA Spring ConferenceManage Subscription | Contact Us
Replete with wizards and widgets, the latest development tool from LANSA can create a collection of Web applications in a matter of minutes.
Written by Chris Smith
The need for interoperability and the growing variety of skill sets developers find they need to create Web applications in an IBM i environment have focused attention again on the benefits LANSA can bring to the development environment. The company has complied with the needs of changing times by introducing a new version of Visual LANSA and opening up the LANSA repository to all programs.
There are more ERP solutions than ever before today, and here we help you pick the right one by describing the major players.
Written by John Ghrist
Enterprise Resource Planning (ERP) is another one of those computer industry terms that seems to have different definitions, depending on who's talking. Wikipedia defines ERP as a system "intended to manage all the information and functions of a business or company from shared data sources." But as many would be quick to point out, how then do we differentiate ERP from such terms as business process management, management information systems, or even management reporting, which arguably all have that same goal?
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Net Prime One Inc. provides the highest level of Information Technology (IT) consulting services in the areas of security, risk management, infrastructure, communications and networking, enterprise architecture and contingency planning.We provide these services to both government and private sectors. Net Prime One's trusted, best-of-breed IT solutions mitigate security and compliance risks, enhance accountability, lower IT costs, reduce turnover, and increase management efficiency.Net Prime One is privately held, with its headquarters located in the province of Ontario, Canada with a strong presence in the province of Quebec, Canada. Net Prime One consultants possess expertise and hands-on experience in most areas of IT consulting including the following:
What We Do