Bloomington, Ill., Nov. 18, 2017 – The Washington University in St. Louis swimming & diving program finished on top of both the men's and women's five team pool of competition in the Illinois Wesleyan University Invitational Saturday evening.
The men finished with the largest lead totaling 179 points to defeat Augustana College (111), Illinois Wesleyan (77), Millikin University (33) and Principia (31). The women finished with a meet-high 186 points as Illinois Wesleyan (124), Augustana (69), Millikin (68) and Principia (17) followed the Bears.
Senior divers Laura Barber and Ryan Nathenson both finished with season-high scores and first place finishes in their respective dives. Barber placed first with a personal-best one-meter mark 420.80 which moves her closer to NCAA qualification and a season-best three-meter mark of 258.00 while Nathenson captured first in the one-meter dive (409.35) and three-meter dive (235.90).
Following the morning diving results, the WashU women were led by sophomore Mollie Seidner and senior Nicole Zanolli. Seidner totaled two first place finishes individually while also pairing up in a first place relay finish. She captured first in the 500 freestyle (5:20.33) with a season-best time and also earned first in the 200 IM (2:16.24).
Seidner was the anchor of the Bears winning 200 medley relay which included Zanolli, sophomore Jenna Wilf and senior Niamh O'Grady. The relay team posted a time of 1:51.17.
Zanolli also earned first place finishes in the 200 freestyle (1:56.17) and the 100 backstroke (1:00.05). First place efforts were also earned by freshman Ella Needler in the 100 butterfly (1:00.46) and sophomore Stephanie Li in the 100 breaststroke (1:09.97).
The men were led by junior Max Cardwell's three first place efforts. He placed first in the 200 freestyle (1:48.27) and the 100 freestyle (49.14). He wrapped up his evening as the anchor on the winning team of the 200 freestyle relay (1:27.99) with junior Andrew Pek, sophomore Jerry Chen and sophomore Chase Van Patten.
Chen and Van Patten also earned first place individual finishes. Chen swam to a first place finish in the 100 breaststroke (1:02.38) while Van Patten's season-best 53.53 time in the 100 butterfly placed him ahead of the competition.
The Bears 200 medley relay squad also swam to first place honors with a time of 1:37.63, which was led by junior Kevin Van Cleave, freshman Matthew Yang, junior Brandon Lum and sophomore Peyton Wilson. The women's 200 freestyle relay (1:40.44) also captured first freshman Katie Cardwell, freshman Sabrina Baxamusa, sophomore Catherine Cowin and junior Maddie Robinson.
WashU returns to the pool Thurs.-Sat., Nov. 30-Dec.2 at the Miami (Ohio) Invitational for the three-day event, while also sending Bears to the Wheaton Invitational Fri.-Sat., Dec. 1-2. Be sure to stay up to date on all your Bears scores and awards by following @WashUBears on Facebook, Twitter and Instagram.


Cool combi-top fram Danish Best Behavoir. It is oversized and has funny details. The inmost part is a regular tank top. Thereafter you put on the next layer which gives the beautiful waterfall effect on the front of the top. On the waterfall there is a nice detail with a seam which looks like many close layers have been sewed together. With the colour mint the blouse are see-through on the back. Both colours are really striking next to the bronzed summer skin. Use the top with jeans and flat shoes.


Capital eg your house, savings, shares.
The burning question for most people is 'what will I get?' or 'what will I owe?'.
There is no magic formula for this and it is not simply a case of arithmetic. Divorce legislation sets out a checklist of things that a court takes into account when asked to make decisions about how to divide up matrimonial assets, income and pensions. In most cases, the court carries out a balancing exercise of different factors and there is often more than one answer - courts work in terms of bands of reasonableness. It is therefore important that you do not assume that information friends or relatives give about their own experience of financial matters will be the right advice for you.
We are experts in this area and can advise you once full and frank disclosure of both parties' financial circumstances have been obtained on what would be a fair financial settlement for you. We take into account all of the relevant financial circumstances and help you negotiate your financial settlement. Where it is not possible to reach an agreement with your partner, we can represent you by robustly pursuing proceedings on your behalf, the objective being obtaining a successful outcome for yourself.
You may want peace of mind and financial protection when entering into marriage and to regulate your financial affairs should you separate. For example, this can relate to inherited wealth or property and assets from a previous marriage. With the Supreme Court ruling about Prenuptial Agreements, couples are likely to increasingly want to protect themselves financially. We can give you expert legal advice about Prenuptial Agreements our you can get a guide as to what difference one could make by using our prenup calculator.
Winston Solicitors LLP provide family mediation services for both individuals and legal professionals across West Yorkshire. Our family mediator Anna Vollans is a fully accredited and experienced family law mediator and collaborative lawyer.


text:
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Defined by Daring
Exclusive stories from makers across America
Published on Monday, January 31, 2011. This article appears in the February/March 2011 issue of American Craft Magazine.
Author Suzanne Smith Arney
Joan Schulze is a master of juxtaposition, pairing and layering images until they're more than the sum of their parts.
Mark Tuschman
Quiltmaker, collage artist, photographer – and that only begins to describe Joan Schulze and the scope of her art.
To look at Joan Schulze's quilts is to see life through the eyes of an explorer. At 74, she feels the same curious anticipation toward a new day that she remembers feeling as a child. "Fearless" is how she describes her young self - a boldness grounded not in arrogance, but in pragmatism. "I always figured I'd be right 50 percent of the time," she says. By 8, she could wend her way solo through Chicago, navigating busy streets and conning her way into the Art Institute. Moving to California at 30, she discovered a more open landscape, filled with light and color.
Schulze flourished in this land of enticing vistas. Everywhere she went she met artists - she recalls being especially inspired by a neighbor who made a painting a day - and she recognized herself as one. Though she'd been sewing for years, she began exploring fabric in the same way she'd explored Chicago: trying anything, making beelines, taking detours. Her compass? "I just made what felt good to me." She was drawn to transparent materials, foreshadowing the sheer silk and two-sided quilts to come; her appliqué work became increasingly complex collage.
Fast-forward to the present, and the journey Schulze launched in 1967 continues undeterred. In addition to the mixed-media quilts for which she is best known, the Sunnyvale, California-based artist also claims in her repertoire collage, photography, altered books, sculpture, and several volumes of poetry. She regularly leads classes, workshops, and lectures around the world. In 2010, the San Jose Museum of Quilts & Textiles organized a 40-year retrospective of her work, "Poetic License: The Art of Joan Schulze." A richly illustrated 168-page book of the same name was published in conjunction with the exhibit, mapping landmarks in a career defined by daring.
From her first explorations of creative needlework, Schulze sought to bring together the techniques and materials that would best express her singular intentions. Besides using floss and fabric, she recycled her dresses and employed paper, pantyhose, currency, plastic, and dryer lint. In an appliqué class she took soon after settling in the Golden State, Schulze experimented with visual and textural effects. She embellished, scrunched, stacked, added, and subtracted. The following year, Abalone, a texture-rich appliqué of cotton, silk, and nylon net, finished with hand-stitching and cutwork, was accepted into a juried exhibition organized by the Peninsula Stitchery Guild.
She always intended her quilts to be seen as artwork. She was persistent in contacting galleries and insistent that her work be judged by the same criteria as paintings. In the mid-1970s she received her first solo exhibition at the Triton Museum in Santa Clara, California, and her first commissions. Fiber artists Jean Ray Laury and Constance Howard became her mentors and lifelong friends.
Schulze tried many approaches to needlework and eventually added photography, printmaking, and dyeing to her skill set. But quilting would become her métier. "Stitching gives me a chance to enjoy all the nuances," she says - "the irregularities of the glue-transfer process, the juxtaposition of images and lines - and a way to emphasize and strengthen the composition while adding texture to the piece."
Composition is where Schulze shines. In preparing the elements of her art, she has altered fabrics through painting, photography, photocopy transfer, and, in recent years, digital technology. She always has her camera on hand; many quilts include transfers of photographs she shot herself. But while a camera objectively records scenes, it is Schulze who chooses the images and juxtapositions for her work. City Woman, 2010, combines a woman's face, foot, and accessories with elegant rooms, flowers, an open book, and an observer.
"Joan's quilts draw on notions of abstraction," scholar Sarah Tucker writes in Poetic License, "to explore and express in visual terms a need to go beyond the confines of material experience - reality - to that of the senses and the imagination."
As an artist, Schulze is fascinated with layers. "Between them lies the opportunity to freeze time, work with complex ideas, and explore another dimension," she says. "When a work is finished, my final question is: Is it more than the sum of its parts?" For Private Dreams of the Writer, 2005-2006, Schulze collaged monoprints, photocopies, printed text, and drawing onto silk. The prints are fragmented, some over-painted and inked, leaving the writer's dreams unarticulated but forming a compositional cohesion. Work on hand is open to reassessment; in 2010 she resized the machine-quilted piece as a 14-by-54-inch scroll.
In Angel Drawings, an ethereal meditation in black thread on silk organza that Schulze made in 2003, machine-stitched lines seem to take form, then dissolve. You can sense a similar reverie in one of her poems: "I sit and spin my thoughts / high up in the air / my shadow goes before me / drifting among floating clouds." Like her quilts, Schulze's poetry is collaged, layered, and subtle.
It's this boundless creative acuity that makes Schulze a sought-after instructor and juror. This year, she'll teach in Australia and New Zealand, as well as at Haystack Mountain School of Crafts and the Pacific Northwest Art School.
But China holds a special allure for her. Since her invitation to the first Fiber Art Biennale in Beijing in 2000, Schulze has participated in every one as an artist, committee member, or juror. She recently returned from her seventh trip to the country, where she was a visiting professor of art at Beijing's Tsinghua University. She was instrumental in bringing the exhibition "Changing Landscapes: Contemporary Chinese Fiber Art" to the San Jose Museum of Quilts & Textiles in 2009. SJMQT has plans to send the retrospective of her work to China in 2012; a Chinese translation by Teresa Huang is already part of the Poetic License book.
While the technical aspects of her work are considerable, Schulze has never avoided challenges or uncharted terrain. Quilting is "the most heroic thing I do in the studio," she says. Skills and central concerns deepen over time; new understandings take shape; inspirations and obstacles lead to breakthroughs. And in opening her discoveries to us, she gives us a larger, multilevel perspective of the world we share.
Suzanne Smith Arney is a freelance writer in Omaha, Nebraska, with a particular interest in fiber and quilts.
Repose, 2004; quilted silk, paper; 24 x 24 in.
In Schulze's hands, familiar playing cards become otherworldly portals. This collage is 'Night Two (8 x 6 in.).
The Good Years, another 8-by-6-inch collage.
Figure F, 2008; collage on paper; 10 x 8 in.
Schulze squares an element into a collage in her newest series, Time to Speak.
She peels off loose paper bit by bit, dampening it before removing more. Schulze is careful not to disturb the images unless she wants a distressed effect.
She sews collaged pieces together before batting, backing, and, finally, quilting.
Figure D, 2009; collage on paper; 10 x 8 in.
The Queen's, another 8-by-6-inch collage.
Touched by Moonlight, 2004; kimono fabric, silk, cotton; 15.25 x 43 in.
For Visitors, 2009, Schulze machine-quilted silk and paper that she altered through collage and a glue-transfer process. The piece is 44 by 84 inches.
Figure C, 2009; collage on paper; 10 x 8 in.
What new material or approach are you planning to explore in 2011?
To celebrate the Renwick's 40th anniversary in 2012, we are mounting a major travelling exhibition, Craft Futures: 40 Under 40, which will showcase
Deeply Felt Creations
In her mixed-media sculptures, Susan Aaron-Taylor recreates the landscape of her dreams.
If you want to understand artist Julie Chen's exquisite books, start by getting your hands on one.
We champion craft.

redpajama_set_name: RedPajamaCommonCrawl


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Marvelous Design For A Second Rate House: Elevation, Section And Plan | Riba Plan Elevation Section Of A House Pic - The image above with the title Marvelous Design For A Second Rate House: Elevation, Section And Plan | Riba Plan Elevation Section Of A House Pic, is part of Plan Elevation Section Of A House picture gallery. Size for this image is 630 × 630, a part of House Plans category and tagged with Section, Elevation, House, Plan, published January 29th, 2019 14:00:23 PM by Edgar. Find or search for images related to "Marvelous Design For A Second Rate House: Elevation, Section And Plan | Riba Plan Elevation Section Of A House Pic" in another posts. Back to: Plan Elevation Section Of A House.


When our council put traffic claiming measures in Browns Lane/Windmill Hill by narrowing the road at intervals along the road I said it would not work, it was an accident waiting to happen and was a waste of taxpayers' money.
Well it looks like I and others were right because Councillor Galliers now has a petition in place for more traffic claiming measures for this area but at least he is asking residents for their ideas.
Well I'll give you an idea: Enforce the law! It is a 30mph limit after all, but for that you will need to spend the money on policing the area instead of more roadworks.
More police in the area would not only cut down on the speeding but would stop the burglaries and even put a stop to idiots messing with cars late at night.
I had my car messed with for a number of months, even causing over £150 worth of damage. The police did not have the resources to catch this person so I had to put CCTV in place to catch him and when I did all the police could do was go round to his house and talk to him.
Now my wife is terminally ill and I cannot afford to have my car in for repair as I need it to take my wife to hospital for treatment. This person refused to pay for the damage even though I have asked him to make the cheque out to Myton Hospice.
Attendance at ward forums is low – many people find them ineffective and a waste of time.
One reason for this is that they are chaired by a councillor of the majority party. Consequently they are used as a way to send the message of how well the council is doing, how great things are and what tremendous progress is being made.
This has the dangerous consequence that the major difficulties the city is facing are likely to be ignored and not faced up to – poverty, poor health, low educational attainment and an economy on its knees.
I'd like to make three suggestions to make ward forums make a more useful contribution to local democracy. The first is an independent Chair – a person competent to run a meeting, with no political affiliation – perhaps a local professional person such as a GP.
Secondly to have the agenda for the next meeting set from the floor, by local citizens, and not by the council.
Thirdly – taking minutes. Currently 'notes' of meetings are taken by an officer employed by the council – who will naturally not have an independent point of view.
Instead minutes could be taken by someone not a council employee, such as a business administration undergraduate, for example.
This is an attempt to make the democratic process more effective.
How wonderful to read that University Hospital is receiving an award from Barcelona for the specialised work that consultant Mr Weickert has done in endocrinology at the hospital.
It's nine years this coming July since the new-build at Walsgrave was opened, with the NHS generally running at a loss and the chaotic parking, bad logistics of traffic arrangements, complaints about the parking charges.
An awful lot of very professional speciality work goes on there, but as people aren't happy in Coventry unless they are moaning and groaning, the first class professionalism and service the consultant physicians have is often forgotten by bad press.
Well done Walsgrave. To secure an achievement of that magnitude needs a privileged place in the hospital so all and sundry can see. Thank you.
I was shocked and saddened to hear Geoff Foster had suffered a stroke (Red Button, March 6).
I knew him from my days as secretary to the manager(s) at Coventry City. A very likeable man with a lovely smile.
Like Lorna, his friend and colleague, I heeded the advice of the F.A.S.T. adverts a couple of years ago and was able to get prompt help for a friend and neighbour.
Get well soon Geoff. You are greatly missed.
To think Coventry God Cakes are being made and sold on the other side of the world in Thailand (Letters, March 7). I wonder if we can get some here in Coventry.


text:
Membrane Transport, Structure, Function, and Biogenesis| Volume 282, ISSUE 21, P15559-15568, May 25, 2007
Flippase Activity Detected with Unlabeled Lipids by Shape Changes of Giant Unilamellar Vesicles*
Andreas Papadopulos
Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Institut für Biologie/Biophysik, Invalidenstrasse 42, D-10115 Berlin, Germany
Stefanie Vehring
Iván López-Montero
Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France
Lara Kutschenko
Martin Stöckl
Philippe F. Devaux
Michael Kozlov
Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, 69978 Tel Aviv, Israel
Thomas Pomorski
To whom correspondence may be addressed. Tel.: 49-30-2093-8830; Fax: 49-30-2093-8585
* This work was supported in part by Deutsche Forschungsgemeinschaft Grant He1928 - 6 (to A. H.) and Po748 - 4 (to T. P.). A grant from the European Community, MRTN-CT-2004-005330 (to A. H., P. F. D., M. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-4.
1 These authors contributed equally.
2 Received support from a Short Term Scientific Mission from COST D22 action on lipid protein interactions and from the Berliner Chancengleichheits-Programm N-318/06.
3 Supported by research grants from the CNRS (UMR 7099) and by Université Paris 7-Denis Diderot.
Open AccessPublished:March 17, 2007DOI:https://doi.org/10.1074/jbc.M604740200
Previous ArticleSelective Amino Acid Substitutions Convert the Creatine Transporter to a γ-Aminobutyric Acid Transporter*
Next ArticleCell Phenotype-specific Down-regulation of Smad3 Involves Decreased Gene Activation as Well as Protein Degradation*
Transbilayer movement of phospholipids in biological membranes is mediated by energy-dependent and energy-independent flippases. Available methods for detection of flippase mediated transversal flip-flop are essentially based on spin-labeled or fluorescent lipid analogues. Here we demonstrate that shape change of giant unilamellar vesicles (GUVs) can be used as a new tool to study the occurrence and time scale of flippase-mediated transbilayer movement of unlabeled phospholipids. Insertion of lipids into the external leaflet created an area difference between the two leaflets that caused the formation of a bud-like structure. Under conditions of negligible flip-flop, the bud was stable. Upon reconstitution of the energy-independent flippase activity of the yeast endoplasmic reticulum into GUVs, the initial bud formation was reversible, and the shapes were recovered. This can be ascribed to a rapid flip-flop leading to relaxation of the monolayer area difference. Theoretical analysis of kinetics of shape changes provides self-consistent determination of the flip-flop rate and further kinetic parameters. Based on that analysis, the half-time of phospholipid flip-flop in the presence of endoplasmic reticulum proteins was found to be on the order of few minutes. In contrast, GUVs reconstituted with influenza virus protein formed stable buds. The results argue for the presence of specific membrane proteins mediating rapid flip-flop.
In lipid bilayers the spontaneous movement of major phospholipids, e.g. of phosphatidylcholine (PC),
The abbreviations used are: PC, phosphatidylcholine; CPM, 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin; ER, endoplasmic reticulum; GUV, giant unilamellar vesicles; LPC, l-α-lysophosphatidylcholine; PE, phosphatidylethanolamine; TE, Triton X-100 extract; RT, room temperature; NBD, 7-nitrobenz-2-oxa-1,3-diazol-4-yl; C6-acyl-PC, 1-acyl-2-hexanoyl-sn-glycero-3-PC; C6-NBD-PC, 1-palmitoyl-2-(NBD-hexanoyl)-sn-glycero-3-PC.
6The abbreviations used are: PC, phosphatidylcholine; CPM, 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin; ER, endoplasmic reticulum; GUV, giant unilamellar vesicles; LPC, l-α-lysophosphatidylcholine; PE, phosphatidylethanolamine; TE, Triton X-100 extract; RT, room temperature; NBD, 7-nitrobenz-2-oxa-1,3-diazol-4-yl; C6-acyl-PC, 1-acyl-2-hexanoyl-sn-glycero-3-PC; C6-NBD-PC, 1-palmitoyl-2-(NBD-hexanoyl)-sn-glycero-3-PC.
between the two monolayers is slow, with half-times on the order of hours or even days. However, lipid topology of cellular membranes results from a continuous bidirectional movement (flip-flop) of lipids between the two leaflets in which specific membrane proteins, so called flippases, play an essential role (
Kornberg R.D.
McConnell H.M.
Holthuis J.C.M.
Levine T.P.
Nat. Rev. Mol. Cell Biol. 2005; 6: 209-220
). Energy-independent flippases allow phospholipids to equilibrate rapidly between the two monolayers, whereas energy-dependent flippases mediate a net transfer of specific phospholipids to one leaflet of the membrane. Candidates for the latter flippase are members of a conserved subfamily of P-type ATPases (
) as well as ATP binding cassette transporters (
Pohl A.
Devaux P.F.
Herrmann A.
Biochim. Biophys. Acta. 2005; 1733: 29-52
In eukaryotes the cytoplasmic leaflet of the endoplasmic reticulum (ER) membrane is the major site of phospholipid biosynthesis. To ensure stable membrane growth, energy-independent flippases mediate rapid, bidirectional, and rather unspecific phospholipid flip-flop with half-times of minutes or less (
Bishop W.R.
Bell R.M.
Cell. 1985; 42: 51-60
Zachowski A.
Buton X.
Morrot G.
Fellmann P.
Seigneuret M.
Marx U.
Lassmann G.
Holzhütter H.G.
Wüstner D.
Müller P.
Höhlig A.
Kubelt J.
Biophys. J. 2000; 78: 2628-2640
). A similar flippase activity was also found in the bacterial inner membrane, where lipid synthesis occurs likewise at the cytoplasmic leaflet (
Hrafnsdóttir S.
Nichols J.W.
Menon A.K.
Techniques to determine transbilayer phospholipid movement as well as the activity of flippases have been critically evaluated (
Hervé P.
Chem. Phys. Lipids. 2002; 116: 115-134
). Spin-labeled and fluorescent lipid analogues have provided much insight into protein-mediated transbilayer dynamics of phospholipids (
). However, the bulky reporter moieties may affect the absolute values of transbilayer lipid movement.
An alternative approach to characterize flip-flop is based on shape changes of deflated, prolate giant unilamellar vesicles (GUVs) (
Mui B.L.
Döbereiner H.G.
Madden T.D.
Cullis P.R.
Biophys J. 1995; 69: 930-941
), which do not require labeled lipid analogues. Shape transitions can be triggered by a very small excess of lipid in one monolayer (
López-Montero I.
Rodriguez N.
Cribier S.
Velez M.
Berndl K.
Käs J.
Lipowsky R.
Sackmann E.
Seifert U.
Europhys. Lett. 1990; 13: 659-664
Farge E.
Biophys. J. 1992; 61: 347-357
Tanaka T.
Sano R.
Yamashita Y.
Yamazaki M.
Langmuir. 2004; 20: 9526-9534
). For example, unlabeled lipids were inserted into the external leaflet of GUVs made of egg-PC (
). Because of the low compressibility of lipid monolayers, the asymmetric lipid supply creates a surface area difference between the two leaflets, which in turn results in formation of a bud-like structure (Fig. 1A). If lipids (e.g. lyso-PC, LPC) that are known to undergo a very slow flip-flop similar to that of egg-PC were added, the bud was stable, whereas if the added lipids (e.g. ceramide) were able to rapidly redistribute between the two membrane leaflets, the initial bud formation was reversible, and the prolate shape was recovered (
). This can be explained by a relaxation of the monolayer area difference due to the transbilayer equilibration of the inserted lipids. The time dependence of shape changes could be used for derivation of the flip-flop rate constant.
FIGURE 1Flip-flop of phospholipids assessed by shape changes of egg-PC GUVs. Insertion of exogenously added phospholipids (e.g. egg-LPC) at t = 0 into the outer leaflet caused budding. In the absence of rapid lipid flip-flop, the bud remained essentially stable (A; see Fig. 2), whereas in the presence of a rapid spontaneous or flippase-mediated lipid flip-flop (B), the bud retracts, and the GUV returned to its original shape (see Fig. 4). At t1 bud formation is observed, whereas recovery of the original shape begins at t2 (see "Results").
Here we demonstrate by reconstitution of yeast ER flippase activity into GUVs that dynamics of GUV shape changes can serve as a tool for quantitative characterization of the lipid transport activity of flippases (Fig. 1B). In this case the characteristic time of the sequential shape transformation of protein-containing GUVs is on the order of flip-flop of spin-labeled and fluorescent phospholipid analogues found previously in microsomes (
). The major advantage of this approach compared with the previous ones is the use of unlabeled lipids, which allows avoiding possible artifacts related to the utilization of lipid analogues with bulky reporter moieties (
EXPERIMENTAL PROCEDURES
Materials—Egg l-α-lysophosphatidylcholine (egg-LPC), egg phosphatidylcholine (egg-PC), egg phosphatidylethanolamine (egg-PE), and dioleoylphosphatidylglycerol (PG) were from Sigma-Aldrich. 1-Acyl-2-hexanoyl-sn-glycero-3-phoshatidylcholine (C6-acyl-PC) was synthesized from egg-PC (
Methods Mol. Biol. 1994; 27: 161-175
). C6-ceramide, C16-ceramide, and galactosylceramide were synthesized according to López-Montero et al. (
). 7-Diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM), BODIPY FLC5-PC and -Cer were purchased from Molecular Probes (Eugene, OR).
Yeast Strains and Culture Conditions—The yeast strain sec61 (MATα ura3-52 leu2-3-112 ade2-10 sec61ts) obtained from J. Holthuis (University Utrecht, The Netherlands) was used. Microsomal membranes containing a green fluorescent protein-tagged transmembrane domain linked to carboxypeptidase yscY (for details, see Ref.
Taxis C.
Hitt R.
Park S.H.
Deak P.M.
Kostova Z.
Wolf D.H.
) were derived from W303-1C Δder3 (MATα ade2-1 ura3-1 his3-11, 15 leu2-3, 112 trp1-1 can1-100 prc1-1, Δder3::HIS3) harboring the plasmid pMA1 (kindly provided by D. Wolf, University Stuttgart, Germany). Strains were grown at 27 °C in liquid YPD medium (1% Bacto yeast extract, 2% Bacto peptone (Difco, 2% glucose). The strain W303-1C Δder3 harboring pMA1 was grown in the absence of uracil in synthetic complete medium with 2% glucose at 27 °C. Cells were harvested in the exponential phase, and growth was monitored by A600.
Microsome Preparation—Microsomes were prepared essentially according to Zinser and Daum (
Zinser E.
Daum G.
Yeast. 1995; 11: 493-536
). Briefly, cells were pretreated with 10 mm dithiothreitol in 100 mm Tris sulfate (pH 9.4) for 10 min, incubated with Zymolyase-100T (ICN Biomedicals, Eschwege, Germany) at 5 mg/g of cells (wet weight) in buffer (20 mm potassium phosphate (pH 7.2), 1.2 m sorbitol) at 30 °C for 30 min, harvested, and then lysed in homogenizing buffer (10 mm Tris-HCl (pH 7.4), 0.6 m sorbitol) containing protease inhibitors (1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin A, 5 mg/ml antipain, 1 mm benzamidine, 1 mm phenylmethylsulfonyl fluoride) using a Dounce homogenizer with a tight-fitting pestle. The cell lysate was centrifuged at 3000 × g for 5 min. The pellet was resuspended in homogenizing buffer, homogenized, and centrifuged as above. Both supernatants were pooled. Membrane pellets from differential centrifugation (9,000 × g for 10 min, 20,000 × g for 30 min, 40,000 × g for 30 min, 100,000 × g for 45-75 min) were screened for organelle marker proteins by immunoblotting. The green fluorescent protein-tagged ER membrane protein became enriched in the 40,000 × g fraction. Pellets of 40,000 × g and 100,000 × g enriched in ER membrane proteins were resuspended in buffer T (10 mm Tris-HCl (pH 7.4), 1 mm EDTA) using a tight fitting Dounce homogenizer and stored at -80 °C. If not stated otherwise, the 100,000 × g fraction was used for further preparation of proteoliposomes.
Preparation of Proteoliposomes—Microsomal fractions (20 mg/ml protein) were solubilized by diluting half with 1.6% (w/v) Triton X-100 (Roche Diagnostics) in buffer T. After incubation for 45 min on ice, insoluble material was removed by centrifugation (177,000 × g, 30 min, 4 °C). The resulting supernatant is designated as Triton extract (TE). For reconstitution, egg-PC in chloroform was dried under nitrogen in a glass tube and then dissolved in buffer TX (10 mm Tris-HCl (pH 7.4), 1 mm EDTA, 0.8% w/v Triton X-100) to a final concentration of 4.5 mm. Various amounts of TE were added to generate proteoliposomes with different lipid/protein ratios. Protein-free liposomes were prepared similarly by substituting buffer for TE. To remove detergent and generate vesicles, 100 mg of SM2 BioBeads (Bio-Rad) per 1 ml of solution were added. After 3-4 h rotating at room temperature (RT), the samples were supplemented with an additional 200 mg/ml of BioBeads and transferred to 4 °C for further 14-16 h of mixing. Turbid suspensions were withdrawn, carefully avoiding collection of any beads, and adjusted to an 8-10-fold excess of buffer T. The vesicles were collected by centrifugation (200,000 × g, 50 min, 4 °C) and resuspended at 9 mm phospholipid using a Dounce homogenizer. Protein recovery in the reconstituted vesicles was about 30-45%; lipid recovery was about 90%. More than 99.9% of the original Triton X-100 was removed, as determined by extraction with four volumes of chloroform/methanol (1/2, v/v) and subsequent measurement of the supernatant at A275.
To label microsomal proteins before reconstitution, 450 μl of TE (0.9 mg protein) were mixed with 20 μl of a 10 mm Me2SO solution of the thiol-reactive fluorescent probe CPM and incubated at 4 °C overnight in the dark. Unbound label was removed by dialysis. Analysis by SDS-PAGE and thin layer chromatography demonstrated that at this temperature only proteins but no membrane lipids became labeled (data not shown).
Virosome Preparation—Virosomes were prepared from influenza viruses (strain X31) essentially as described for proteoliposomes. A mixture of virosomal TE, Triton X-100-solubilized egg-PC, and egg-PE (14.75/0.275, w/w) was used for reconstitution at lipid/protein ratio of 20 (w/w). To visualize reconstitution of viral proteins, viruses were preincubated for 2 h at RT in the dark with CPM, added from 5 mm Me2SO stock to molar excess of 10 over hemagglutinin. To remove uncoupled CPM, labeled viruses were washed twice in phosphate-buffered saline (pH 7.4) and harvested by centrifugation at 45,000 × g. SDS-PAGE analysis revealed that only influenza virus hemagglutinin was labeled (data not shown).
Preparation of Giant Unilamellar Vesicles—GUVs were generated from proteoliposomes by the electroformation technique in a chamber made of indium-tin-oxide-coated glass slides at RT (
Angelova M.
Soleau S.
Meleard P.
Faucon J.F.
Bothorel P.
Prog. Colloid. Polym. Sci. 1992; 89: 127-131
Mathivet L.
Girard P.
Pécréaux J.
Lenoir G.
Falson P.
Rigaud J-L.
Bassereau P.
). The oscillating electric field that was applied only during GUV formation did not affect the activity of membrane proteins (
). For GUVs consisting only of lipids, 70 μl of lipid solution in chloroform (0.25 mg/ml) were deposited in small droplets onto each slide. Subsequently, solvent was evaporated in a desiccator (10 mbar) for 60 min at RT. To prepare GUVs with reconstituted proteins according to Girard et al. (
), proteoliposomes were diluted with distilled water to 0.4 - 0.8 mg/ml lipid, and 25-50 μl of this suspension was deposited onto each glass slide in small droplets. To avoid denaturation, proteoliposomes were dehydrated only partially by placing glass slides in a sealed chamber containing a saturated NaCl solution. After overnight incubation a film had formed on the glass slides.
Subsequently, the chamber was assembled by sealing both glass slides with silicon paste. The sucrose solution (250 mm sucrose, 0.02% NaN3) was injected with a syringe (about 1 ml) through a micropore filter immediately before connecting the completely sealed chamber to the generator. The voltage of the applied AC-field was increased stepwise every 6 min from 20 mV up to 1.1 V while continuously increasing the frequency from 4 to 10 Hz within the first minute. The AC field was applied for 3-12 h. To complete the procedure voltage was raised to 1.3 V, and the frequency was lowered to 4 Hz for 1 h. The chamber was then stored in a refrigerator at 4 °C.
Observation and Analysis of GUV Shape Changes—Spherical GUVs cannot undergo shape changes due to the minimized surface to volume ratio. Therefore, deflated vesicles appropriate for shape change studies (
) were achieved osmotically by transferring 10-50 μl of GUVs into 150 μl of a glucose solution (275 mm glucose, 0.02% Na3) on a microscope slide and allowing water to evaporate for 30-60 min at room temperature. To prolate GUVs, lipids that are known to incorporate rapidly into lipid bilayers were added. Typically, 10 μl of 0.1 mm lipid (typically egg-LPC) in glucose solution was injected (representing time zero in the shape changes analysis). The time points t1 (begin of budding transition) and t2 (begin of bud retraction) were measured by microscopic observation of GUV shapes for each experiment (see "Results"). All experiments were performed at room temperature. To determine the amount of lipid incorporated into GUVs, we used radioactive l-lyso-3-phosphatidylcholine,1-[1-14C]palmitoyl (CFA633, Amersham Biosciences). 50 μl of GUVs were transferred to a microscope slide, and 150 μl of a glucose solution (275 mm glucose, 0.02% NaN3) was added. After GUVs settled, radioactive lipids were added, and the suspension was incubated for up to 2 h. Subsequently, aliquots from the supernatant were taken, and radioactivity was measured.
Miscellaneous—Protein content was quantified after TCA precipitation using micro-BCA Protein Assay reagent (Pierce) and BSA as a standard. Phospholipid content was determined by extracting lipids according to Bligh and Dyer (
Bligh E.G.
Dyer W.J.
Can. J. Med. Sci. 1959; 37: 911-917
) and measuring the amount of phospholipid phosphorus (
Rouser G.
Fleischer S.
Lipids. 1970; 5: 494-496
Shape Changes of Protein-free GUVs—Insertion of unlabeled lipids such as egg-LPC, C6-acyl-PC, or C6-ceramide into the external leaflet of GUVs made of egg-PC resulted in formation of a bud-like structure (Figs. 1A and 2). If egg-LPC or C6-acyl-PC, which are known to undergo a very slow flip-flop like egg-PC, was added, the bud was stable (Fig. 2). Whereas if lipids were added that rapidly redistribute between the two membrane leaflets, e.g. ceramide, the initial bud was reversible, and the prolate shape was recovered (
). This can be explained by a decline in the monolayer area difference due to the transbilayer equilibration of inserted lipids. Bud formation was also reversible when egg-LPC was added to egg-PC GUVs containing already a lipid that flips rapidly, such as the long chain C16-ceramide (5 mol %) (not shown). When ceramide was replaced by the slow flipping galactosylceramide, the prolate shape was not recovered after 1 h. The experiments with ceramide and egg-LPC prove that the transversal diffusion of an unlabeled lipid with a fast flip-flop rate can be measured in GUVs even disregarding the flipping ability of the added lipid. However, this conclusion may not be generalized. Although cholesterol is known to flip rapidly across the lipid bilayer (
Leventis R.
Silvius J.R.
), Mathivet et al. (
) did not observe retraction of the initial bud formation when egg-LPC was added to egg-PC GUVs containing up to 23 mol % of cholesterol. A possible explanation could be that transbilayer redistribution of cholesterol cannot compensate for area asymmetry due its specific molecular properties, e.g. it is known that cholesterol cannot form bilayers.
FIGURE 2Shape transition of egg-PC GUVs upon the addition of egg-LPC. Insertion of egg-LPC into the outer leaflet caused the formation of a stable bud that did not retract in the time course of the experiment (for details, see "Experimental Procedures").
To substantiate our conclusions on lipid translocation drawn from GUV shapes, we chose further processes exploiting microscopic visualization of GUV behavior. First, Cullis and co-workers (Ref.
Hope M.J.
Redelmeier T.E.
Wong K.F.
Rodrigueza W.
and publications cited therein) demonstrated that pH gradients influence the distribution of charged phospholipids as phosphatidylglycerol (PG) across the membrane of unilamellar 100-nm vesicles. Using various assays they could prove that PG flips rapidly in its neutral form, whereas it is sequestered within one membrane leaflet in the case of deprotonation in contact with basic environment. Thereby, arising transversal asymmetry could clearly be related to membrane curvature and molecule balance between the leaflets in GUVs (
). When prolate GUVs from egg-PC containing 1 mol % of dioleoyl-PG are exposed to an increased pH (from pH 7 to 9) in the surrounding medium by the addition of NaOH, spontaneous migration of PG from the inner leaflet (pH 7) to the exterior (pH 9) results in relative immobilization due to their acquired head-group charge. The excess molecules account for an area imbalance that is apparent from an instantaneous budding transition (Fig. 3A). The connecting neck is progressively elongated during the following minutes, eventually forming a tether with a highly mobile daughter vesicle (see the change of focus to resolve both structures in last images of series). Control GUVs from egg-PC maintain their prolate shapes for more than 10 min (not shown). By a second independent approach we proved directly that shape changes indeed correlate with the transbilayer distribution of externally added lipids using BODIPY-labeled PC and Cer (see supplemental Fig. 1). Quantification of the fluorescent fraction on the inner surface of the membrane, as determined by protection from trypan blue quenching, is displayed in Fig. 3B. BODIPY-PC proves to be confined essentially to the outer leaflet, whereas the distribution of the Cer analogue resembles that of GUVs prepared with a symmetric BODIPY-PC distribution between both leaflets. We, therefore, conclude that lipid distribution affects the membrane curvature of GUVs, and its dynamics can be reliably visualized as shape changes.
FIGURE 3Shape changes of GUVs correlate with transbilayer redistribution of lipids. A, shape changes with pH-induced flip-flop of phosphatidylglycerol. GUVs were prepared from egg-PC containing 1 mol % dioleoylphosphatidylglycerol. Shown is prolate GUV at pH 7 (a) subjected to pH increase (pH 9) by adding ∼10 μl of NaOH (0.5 n) to ∼400 μl of neutral glucose solution in the observation chamber (b). Phase contrast images are taken with respect to time of alkaline addition (0′00″); the bar corresponds to 10 μm. B, assessment of transbilayer distribution of BODIPY-PC and BODIPY-Cer after shape transitions. BODIPY-PC or -Cer were added to GUVs and incubated for 30 and 10 min, respectively. Subsequently, fluorescence intensities in the absence and presence of the quencher trypan blue (0.0075% w/v) were measured (as described in the supplemental data). Intensity in the absence of trypan blue was set to 1. For comparison, results for GUVs symmetrically labeled with BODIPY-PC are shown (BODIPY-PC*). Data shown are the means ± S.E. of four determinations. Note that fluorescence intensities in the presence of trypan blue do not correlate in a quantitative manner with transbilayer distribution of BODIPY-lipids. For that, higher concentrations of the quencher would be necessary. However, such concentrations affect the stability of the GUVs.
Shape Changes of GUVs with Reconstituted ER Proteins—The flippase activity of yeast ER was first reconstituted into proteoliposomes as already described previously for rat liver (
) and yeast ER (
Nicolson T.
Mayinger P.
FEBS Lett. 2000; 476: 277-281
). Applying established assays for measuring transbilayer movement (see Ref.
and references therein) confirmed protein-mediated flip-flop of fluorescent phospholipid analogues such as 1-palmitoyl-2-(NBD-hexanoyl)-sn-glycero-3-phosphocholine (C6-NBD-PC) being on the order of a minute.
S. Vehring, A. Herrmann, and T. Pomorski, unpublished data.
Subsequently, GUVs were prepared from such proteoliposomes. No significant difference between the size of protein-free and protein-containing GUVs was found (not shown).
When egg-LPC or C6-acyl-PC was added, reconstituted GUVs budded within the first 5 min after injection of lipids and subsequently regained the prolate shape within 5-10 min (Fig. 4). We conclude that the reconstituted ER flippase activity triggers the rapid flip-flop that diminishes the surface area difference between the two leaflets. To demonstrate that GUVs undergoing reversible shape changes contain ER proteins, we reconstituted the ER fraction from a yeast strain expressing an ER resident, green fluorescent protein-tagged membrane protein as well as prelabeled TE (supplemental Figs. 3 and 4).
FIGURE 4Shape transition of GUVs prepared from proteoliposomes containing microsomal membrane proteins (100,000 × g fraction). At time t = 0, egg-LPC was added. GUVs were visualized by differential interference contrast at RT. The lipid/protein ratio was 35/1 (w/w). The budding transition was observed at t1 = 3:25 min. Recovery of the prolate shape started at t2 = 10:35 min.
Shape Changes of GUVs Reconstituted with Influenza Virus Protein—To address whether the flippase activity is a property of specific membrane proteins (
Kol M.A.
van Dalen A.
de Kroon A.I.
de Kruijff B.
), we studied shape changes of GUVs prepared from reconstituted influenza virosomes. Upon the addition of egg-LPC, GUVs formed stable buds (Fig. 5). This indicates that no significant flip-flop has occurred compensating for the surface imbalance between the two leaflets. Hence, the presence of reconstituted membrane proteins per se is not sufficient to trigger rapid flip-flop. These results argue for the presence of specific proteins with a flippase activity in the ER in agreement with previous studies (
Watkins III, W.E.
Curr. Biol. 2000; 10: 241-252
). Notably, the absence of a rapid flip-flop in viral hemagglutinin containing GUVs rules out flip-flop favoring artifacts due to influence of the electric field on proteins during vesicle formation.
FIGURE 5Shape transition of GUVs prepared from virosomes containing influenza hemagglutinin. Viral proteins were reconstituted in proteoliposomes containing egg-PC and egg-PE at a lipid/protein ratio of 20/1 (w/w). At time t = 0, egg-LPC was added. GUVs were visualized by differential interference contrast microscopy at RT.
Determination of Lipid Exchange Rates—To estimate the rates of lipid exchange mediated by flippase activity between membrane monolayers, we used the experimentally observed dynamics of shape changes upon lipid insertion into GUV membranes. For that, the relevant data from experiments are the time points t1 and t2 taken at definite onset of formation and disappearance, respectively, of a spherical bud, both measured from the moment of lipid addition to the aqueous solution surrounding the GUVs.
The GUV shapes are governed to a great extent by the relation of their membrane monolayers (
), where the area asymmetry η is defined as the ratio of area difference ΔA between the monolayers to the area A of the bilayer mid surface: η =ΔA/A. Budding of a prolate GUV has been established to occur when the monolayer area asymmetry exceeds 0.1%, i.e. η ≅ 0.001 (
). Accordingly, t1 and t2 correspond to η ≅ 0.001.
Our analysis includes three sequential tasks; (i) to model theoretically the kinetics of redistribution of exogenously added lipids between the aqueous solution and the GUV membranes; (ii) to express the time of reaching an area asymmetry of 0.1% through the kinetic parameters of the system; (iii) to determine these parameters based on the experimentally measured values of t1 and t2. Within the same kinetic scheme, the present analysis takes into account transbilayer flip-flop as well as lipid exchange between the membrane and the aqueous solutions inside and outside of the vesicle. This provides for self-consistent determination of all kinetic constants exclusively based on the available measurements with unlabeled lipids. In this regard our model advances the one recently proposed for evaluation of the flip-flop rate on the basis of GUV shape changes (
The detailed kinetic scheme describing our system is presented and analyzed in the Appendix. Characterizing the time dependence of the area asymmetry η, the major parameter is the rate km of lipid flip-flop between both monolayers. Determination of km requires knowledge of three other system parameters related to the egg-LPC exchange rates between the membrane and the aqueous solution (see "Appendix").
To illustrate quantitative determination of km, the time points t1 and t2 were taken from microscopic observations presented in Fig. 4 (see images taken for t1 and t2). With 3:25 min (t1) and 10:35 min (t2) we obtained values of 0.3 min-1 for km and 2.3 min for the corresponding half-time, respectively.
In Fig. 6 km is plotted versus the lipid/protein ratio of the vesicle membrane. Despite the considerable scattering, it displays apparent increase of flip-flop with protein content. These values are on the order of those that have been measured for flip-flop of fluorescently labeled phospholipid analogues in the ER of eukaryotic cells with lipid/protein relations on the order of 0.03 (w/w) (see "Discussion").
FIGURE 6The rate constant of flip-flop, km, of lipid in GUVs reconstituted from proteoliposomes containing different amounts of microsomal membrane proteins. km was calculated according to the described model. One way analysis of variance revealed a significant influence of the lipid/protein ratio on km at a significance level of α = 0.1 (FExperiment = 4.041 > F (3, 6, 0.01) = 3.29; F = 4.041 corresponds to α = 0.069).
From the images shown in Fig. 4, the accuracy of determination t1 and t2 is on the order of 10 s. Variation of this order has only a very minor influence on calculation of km (see Table 1 and supplemental data). In this regard, an accuracy of ≥1 min would cause a variation of km of ≥20% (see the three examples at the bottom of Table 1 and supplemental data).
TABLE 1Shape transition times and estimated rate constants for reconstituted GUVs with varying lipid/protein ratio (L/P (w/w)) The times of formation, t1, and disappearance, t2, respectively, of a spherical bud were measured experimentally as described (at t = 0 lipids were added to the aqueous solution surrounding the GUVs). The fitted parameters are: J, lipid flux from surrounding solution into the outer monolayer; kw, rate constant of the egg-LPC transfer from the membrane monolayers to the surrounding solution; km, rate constant of the intermonolayer lipid exchange; kc, rate constant of egg-LPC transfer from the internal aqueous solution into the inner monolayer; b, scaling factor proportional to A/V, characterizing the linear dimension of the vesicle; L/P, the lipid/protein ratio (w/w).
L/P ratio
Ja/kw
min min min-1 min-1 min-1
8 01:43 02:02 0.033 0.03 1.150 0.028
11 02:18 07:12 0.033 0.03 0.830 0.015
The present study demonstrates that GUVs and their shape changes provide a suitable tool to characterize protein-mediated lipid transport across the bilayer. We have shown that the flippase activity of yeast ER enabling rapid phospholipid translocation can be reconstituted in GUVs. The shape change assay independently supports previous studies showing that the spontaneous translocation rates of lyso-PC (
De Kruyff B.
van den Besselaar A.M.
van Deenen L.L.
Biochim. Biophys. Acta. 1977; 465: 443-453
Bhamidipati S.P.
Hamilton J.A.
) and ceramide (
Eckford P.D.W.
Sharom F.
Biochem. J. 2005; 389: 517-526
) differ distinguishably. Therefore, the assay has been applied to flippase protein containing GUVs. The flippase activity was inferred from the reversibility of bud formation triggered by the insertion of added lipids into the external leaflet of prolate GUVs. The half-time of phospholipid flip-flop deduced from the recovery of the original prolate shape was on the order of few minutes, which is indeed very similar to half-times that have been measured by a back exchange assay for the flippase-mediated translocation of fluorescent phospholipid analogues in the ER of eukaryotic cells and in the inner plasma membrane of bacteria. For instance, Marx et al. (
) reported for flip-flop of the fluorescently short chain-labeled phospholipid analogues C6-NBD-PC and C6-NBD-PE in rat liver microsomes half-times of about 1.5 and 2.5 min, respectively. In the same order is the half-time of C6-NBD-PC in microsomes of yeast7 as used here for preparation of GUVs. Likewise, in inner membrane vesicles from Escherichia coli, the half-time for the transbilayer movement of C6-NBD-PE was between 1 and 3 min (
). Although in the latter studies the fast flip-flop could only be probed by labeled analogues, assessment by shape changes of GUVs allows avoiding bulky reporter moieties and can, therefore, be applied to lipids resembling endogenous molecules much better. Moreover, the physicochemical conditions at GUV membranes, which are disposed to flexible reaction to a small molecular excess, offer investigation of lipid translocation with minimal perturbation of the bilayer.
Flippase activity in the ER microsomes and inner membrane of bacteria is rather unspecific and triggers a fast flip-flop of various (phospho)lipid analogues (
Killian J.A.
Vishwakarma R.A.
Vehring S.
Mehta A.
Sinha A.
Pomorski T.
Org. Biomol. Chem. 2005; 3: 1275-1283
). Therefore, we cannot discriminate between flip-flop of exogenously added lipids (egg-LPC) and intrinsic lipids as additionally indicated by results with GUVs containing rapidly flipping ceramide. Typical flip-flop of LPC is very slow and not substantially different from that of phospholipids bearing two long fatty acid chains (
). Thus, we surmise that flip-flop of all lipids present in GUVs is mediated by the flippase.
Flippase Versus Membrane-spanning Helices Induced Translocation of Phospholipids—Transbilayer movement in biogenic membranes as eukaryotic ER or prokaryotic plasma membrane is fast, with flip-flop half-times on the order of minutes or less. Kol et al. (
) found that certain single α-helical membrane-spanning peptides promote transbilayer movement of some phospholipid classes in synthetic membranes, suggesting that lipid flip-flop proceeds in the absence of a dedicated flippase.
Our study clearly demonstrates that the presence of membrane proteins in GUVs per se is not sufficient to facilitate flip-flop of phospholipids. As shown for reconstituted influenza virus hemagglutinin, which exists as a trimer representing three transmembrane domains, the bud formation in GUVs by externally inserted egg-LPC was irreversible, indicating the absence of a rapid flip-flop. Thus, our results argue for the presence of specific proteins (flippases) facilitating a fast transbilayer movement of lipids in the ER. This conclusion supports recent studies on the ER from rat liver cells suggesting the existence of dedicated flippases. In an attempt to identify the flippase(s) of microsomal membranes, Menon et al. (
) reconstituted protein fractions separated either on a glycerol gradient or by anion exchange chromatography into proteoliposomes. This approach yielded protein pools of enriched flip-flop activity, suggesting that specific proteins are responsible for the fast lipid movement in the ER.
Although our study and others suggest the presence of flippases, it does not preclude that membrane spanning α-helices may promote transbilayer movement of lipids as found by Kol et al. (
). However, flip-flop half-times reported therein were significantly longer in comparison to those observed for biogenic membranes in native state as well as fractionated reconstituted into proteoliposomes.
Our study and previous efforts (
) have shown that the flippase activity can be extracted by Triton X-100. Under our conditions, about 10% of ER proteins are solubilized from yeast ER, of which about 40% could be reconstituted. Recently, we have studied the flippase activity in reconstituted ER proteoliposomes (for preparation, see "Experimental Procedures") as a function of the protein to lipid ratio using the fluorescent lipid analogue C6-NBD-PC.7 Based on the obtained dose-response curve and assuming that the inflection point toward saturation reflects the situation where every vesicle would be equipped with at least one flippase, we deduced the abundance of functional flippases as follows: (i) the average proteoliposome diameter of 200 nm (electron micrographs7) gives with a cross-sectional area of 0.6 nm2 for PC 1.4 × 1018 vesicles/mol of phospholipid; (ii) the obtained inflection at about 8 g/mol gives with (i) 5.7 × 10-18 g of protein/vesicle; (iii) with an average molecular mass of 50 kDa for ER proteins (Ref.
Quinn P.
Griffiths G.
Warren G.
J. Cell Biol. 1984; 98: 2142-2147
),7 the initial assumption finally results in a mass proportion for flippase protein of 1.5% of ER extract reconstituted in GUVs. Very likely, flippase proteins constitute a minor fraction of ER proteins. Although the lipid/protein ratio in yeast microsomes (∼0.03 w/w) is about 2 orders of magnitude lower compared with that of proteoliposomes and GUVs (see Table 1 and Fig. 6), the rapid flip-flop in GUVs indicates that the majority of flippase proteins if not all could be recovered in GUVs. Because the molecular identity of flippase proteins is not yet known, we cannot provide an estimate of how many flippase proteins have been reconstituted into GUVs and to which extent this reconstituted system corresponds to native ER membranes regarding this condition.
Application of GUVs to Energy-dependent Lipid Flippases—The present approach should also allow investigation of energy-dependent flippases. Although flippases are very likely reconstituted to a similar extent in both opposing directions, unidirectional lipid transport can be ensured by allowing ATP to access only one membrane leaflet. Characterizing the activity of energy-dependent flippases would not require the addition of lipids. Lipid species to be transported can already be incorporated during GUV assembly. Because of the unidirectional fashion of oriented lipid transport, area differences would be created leading to a shape change. This is different from the visualization of energy-independent flippase activity in GUVs where shape changes have to be triggered first, e.g. by supplementing the external leaflet with additional lipids.
Kinetic Analysis of LPC Partitioning and Transmonolayer Area Asymmetry—Upon the addition of lipid, e.g. LPC, to the aqueous solution surrounding a GUV, lipids partition between the solution and the outer monolayer of the GUV membrane. In the presence of flippase activity, lipid redistribution occurs between the outer and the inner monolayer. And finally, LPC partitions between the inner membrane monolayer and the inner aqueous volume of the vesicle.
We characterize the system by the surface density of LPC in the outer and inner monolayer denoted by X and Y, respectively, the LPC aqueous concentration in the outer solution close to the vesicle membrane, c0, and the LPC concentration in the internal volume of the vesicle, cin. Before insertion of LPC, the areas of the outer, Aout, and inner, Ain, monolayers of GUV are equal, Aout = Ain = A. In the course of LPC partitioning, these areas become
Aout=A⋅(1+aX)
(Eq. 1)
Ain=A(1+aY),
where a is the LPC molecular area. The emerging monolayer area asymmetry is
η=Aout-AinA=a(X-Y).
We assume that the monolayer area asymmetry remains small, |η| << 1, and in all the equations below, we account only contributions of the first non-vanishing order in η.
To describe the entire redistribution process we consider six lipid fluxes illustrated in Fig. 7A; (i) from the outer solution into the outer membrane monolayer,
J1=J=kcc0,
where c0 is the LPC aqueous concentration in the vicinity of the membrane surface, and kc is the water-membrane exchange rate constant; (ii) from the outer monolayer back to the surrounding aqueous solution,
J2=kwX,
where kw is the membrane-water exchange rate constant; (iii) from the outer to the inner membrane monolayer,
J3=kmX,
where kw is the rate constant of lipid exchange between the monolayers; (iv) from the inner back to the outer monolayer,
J4=kmY,
(v) from the inner membrane monolayer to the inner aqueous solution of the vesicle,
J5=kwY,
(vi) from the inner solution back to the inner membrane monolayer,
J6=kccin,
where cin is the inner aqueous concentration of LPC next to the membrane. The rate constants kw, kc, and km and their units are defined according to the standard Langmuir approach to description of molecular exchange between a surface and a volume phase or between two surfaces (
Alberty R.A.
Silbey R.J.
Physical Chemistry.
6th Ed. John Wiley & Sons, Inc., New York1983: 275-296
Adamson A.W.
Physical Chemistry of Surfaces.
3rd Ed. John Wiley & Sons, Inc., New York1990: 548-634
FIGURE 7Model for theoretical determination of lipid exchange rates. A, model. Dependence of monolayer area asymmetry η =ΔA/A (in %) upon insertion of exogenously added lipids into the outer leaflet (t = 0) in the presence (B) and absence (C) of flip-flop. GUV budding has been established to occur when η equals ∼0.1% (η ≈ 0.001) as indicated in B. For details, see "Results."
Equations 2, 3, 4, 5 do not account for the influence of elastic stresses on the lipid fluxes, which can emerge within the membrane monolayers as a result of the monolayer area asymmetry. Estimations show that for realistic values of η, the elastic stress effects provide negligible corrections to Equations 2, 3, 4, 5. Moreover, we describe the lipid fluxes between the monolayers, J3 and J4, by the same rate constant km. This implies that the rate constants are not affected by monolayer bending, which can be justified for the large membranes of GUVs but may be invalid for small lipid vesicles.
The redistribution between the various compartments results in time evolution of the LPC concentrations within the monolayers, X and Y, and in the aqueous compartments, c0 and cin. In our system the outer aqueous volume is very large compared with the volume enclosed by GUVs. Therefore, only a small fraction of the total lipid will be incorporated into the vesicles, meaning that the change of the outer aqueous concentration of LPC can be considered as a constant, c0 = constant. Indeed, by using radioactive l-lyso-3-phosphatidylcholine, 1-[1-14C]palmitoyl (see "Experimental Procedures"), we found that after 2 h of incubation of GUVs, only 11% of added lipids were incorporated. A similar value can be estimated on the basis of the water/lipid partition coefficient of lysolecithin (monopalmitoyl), which is Kp ≈ 0.5 × 10-6 (
Brown S.D.
Baker B.L.
Bell J.D.
). Depending on the amount of GUVs (see "Experimental Procedures"), between 13 and 4% of lipids will be incorporated.
The dependence on time of the three other concentrations, X, Y, and cin, is determined by the following equations.
dXdt=J1-J2-J3+J4=J-kwX-kmX+kmY,
(Eq. 10)
dYdt=J3-J4-J5+J6=kmX-kmY+kwY+kccin.
To obtain the equation for the time dependence of the LPC concentration in the lumen of vesicles, cin, we have to take into account that variation of the total number of LPC molecules in the vesicle lumen, Vcin, is determined by the balance between the total number of molecules entering and leaving the lumen, AJ5 and AJ6, respectively, per unit time (recall, that V and A are the vesicle volume and the area of the inner monolayer, respectively; the fluxes J5 and J6 are determined as the number of LPC molecules exchanged per unit time and unit monolayer area). The resulting equation is,
dcindt=(J5-J6)b=(kwY-kccin)b.
where b = A/V has the unit of inverse length to account for the fact that molecules (LPC) exchange between a volume and an area (monolayer) characterized by concentration and surface density. Solution of this system of equations has to take into account the initial concentration of LPC in the membrane layers and the internal vesicle volume, respectively, to be zero: X(t = 0) = 0, Y(t = 0) = 0, cm(0) = 0.
We consider two types of solution of Equations 10, 11, 12 as relevant for our experimental setup. In a bilayer containing flippases, the rate of lipid exchange between the monolayers has to be high compared with the off rates of LPC from the membrane either to the aqueous medium or to the vesicle lumen, km >> kw and km >> kcb, respectively. The time evolution of the monolayer area asymmetry is given in this case by
η(t)=Ja2km((1+kcbkw)e-kcb2t-(1+kcb2kw)e-2kmt-(kcb2kw)e(-kw-kcb2)t)
In the absence of flippases, the transfer of LPC between the monolayers is very slow, km << kw, and effectively LPC partitions only between the external solution and the outer membrane monolayer. Then the related time evolution of the area asymmetry is given by
η(t)=Jakm(1-e-kwt).
Determination of Kinetic Parameters—Time behavior of the monolayer area asymmetry, η, in the presence of flippases given by Equation 13 has a non-monotonic character, as illustrated in Fig. 7B. The initial increase of η corresponds to redistribution of egg-LPC between the external solution and the membrane monolayers, whereas the decrease of η with time results from egg-LPC partitioning into the internal vesicle volume and equilibration throughout the whole system. The time points of bud formation, t1, and decay, t2, are represented by intercepts of the curve η(t) with the line η = 0.001 (Fig. 7B). Fitting of these intercept points to the experimental values of t1 and t2 gives two equations for the parameter determination: η(t1) = 0.001 and η(t2) = 0.001.
In the absence of flippase activity (Equation 14) the area asymmetry dynamics follow a monotonous increase (Fig. 7C). In this case the first equation of η(t) describes the intercept of the curve η(t) with η = 0.001, and the second equation accounts for the known maximal amount of lipid incorporation, which in this case (egg-LPC) has been already determined to be about 3% of total lipid (
Parameter values obtained from fitting the data of different experiments are presented in Table 1. The parameters kcb and km were determined by trial-fitting of the curve η(t) (Equation 13) to the experimentally measured times t1 and t2. The fitting was performed by varying km and kcb at fixed values of parameters J and kw (Equation 13). The values of J and kw were determined from experiments on egg-LPC insertion into the outer monolayer of GUV in the absence of flippases and, hence, under conditions of negligible trans-monolayer lipid exchange. In this case the number of lipid molecules incorporated into the external monolayer related to the total number of the monolayer lipids, ΔNout/Nout, changes with time according to Equation 14. The coefficient for the resulting expression, Ja/kw, represents the maximal possible value of ΔNout/Nout. Taking into account that for the concentrations used in our experiments the maximal value of ΔNout/Nout is 0.03 (
), we obtain Ja/kw = 0.03. Furthermore, experiments in the absence of flippases showed that bud formation corresponding to incorporation of 0.001 of total lipids (
) occurred about 1 min after the addition of lipids to the solution (Fig. 2). Inserting this value together with Ja/kw ≈ 0.03 in the expression for ΔNout/Nout, we obtain Ja = 0.001 min-1, and kw = 0.03 min-1.
All fitting procedures were only successful for values of km and kcb in a very narrow range, supporting the robustness of the fitting results. However, for some experiments fitting could not be achieved by using Ja = 0.001 min-1, and kw = 0.03 min-1. In those cases the value for J was taken as a third fitting parameter.
Note that the dispersion range of the fitted values of kcb is rather large, with values differing by a factor of 10. This can be ascribed to the experimental setup in which the exact amount of membranes and, in particular, the total lipid concentration is not well controlled and may vary considerably between different experiments by the following reasons. Added lipids might stick to the glass bottom of the dish used for experiments, which reduces the effective lipid concentration. The effective egg-LPC concentration on the vesicle membrane might also be influenced by uncontrolled kinetic restrictions since the egg-LPC solution was not stirred.
We estimated the sensitivity of the fitted values of km and kcb with respect to variation of J and kw. Varying J and kw within a range for which the experimental values of t1 and t2 could still be matched, the order of magnitude of the fitted parameters km and kcb did not change (see Table 2).
TABLE 2Sensitivity of kw and kcb with respect to variation of kw (fixed Ja) and Ja (fixed kw) for t1 = 3:25 min, t2 = 10:35 min (see Fig. 4) The fitted parameters are: J, lipid flux from surrounding solution into the outer monolayer; kw, rate constant of the egg-LPC transfer from the membrane monolayers to the surrounding solution; km, rate constant of the intermonolayer lipid exchange; kc, rate constant of egg-LPC transfer from the internal aqueous solution into the inner monolayer; b, scaling factor proportional to A/V, characterizing the linear dimension of the vesicle.
min-1 min-1 min-1 min-1
0.001 0.01 0.713 0.003
0.001 0.05 0.575 0.0063
0.0006 0.03 0.45 0.012
We gratefully acknowledge Thomas Korte for excellent assistance in fluorescence microscopy, Patricia Bassereau for introduction into protein reconstitution, Bärbel Hillebrecht for help with virus preparation, and Dieter Wolf and Joost Holthuis for generously providing yeast strains and plasmid. Lara Kutschenko (University of Bonn) was a supported by the Studienstiftung des Deutschen Volkes.
Received in revised form: March 16, 2007
DOI: https://doi.org/10.1074/jbc.M604740200


text:
package com.idp.web.generator.controller;

import java.util.Date;
import java.util.List;

import javax.annotation.Resource;
import javax.servlet.http.HttpServletRequest;

import org.apache.log4j.Logger;
import org.springframework.stereotype.Controller;
import org.springframework.web.bind.annotation.RequestMapping;
import org.springframework.web.bind.annotation.ResponseBody;

import com.idp.common.base.BaseController;
import com.idp.common.persistence.Page;
import com.idp.web.generator.entity.CgDataSource;
import com.idp.web.generator.entity.CgTableConfig;
import com.idp.web.generator.service.CgDataSourceService;
import com.idp.web.generator.service.CgTableConfigService;
import com.idp.web.generator.util.ReadTableUtils;

import net.sf.json.JSONObject;

@Controller
@RequestMapping("/tableConfig")
public class CgTableConfigController extends BaseController {

	private static Logger logger = Logger.getLogger(CgTableConfigController.class);

	@Resource
	private CgDataSourceService cgDataSourceService;
	@Resource
	private CgTableConfigService cgTableConfigService;

	@RequestMapping("/init")
	public String init(){

		return "generator/table/tableConfigSearch";
	}

	@RequestMapping("/list")
	public String list(CgTableConfig tableConfig,Page<CgTableConfig> page,HttpServletRequest request){

		request.setAttribute("page", cgTableConfigService.findByPage(tableConfig, page));

		return "generator/table/tableConfigList";
	}

	@RequestMapping("/tableInit")
	public String tableInit(HttpServletRequest request){

		try {

			List<CgDataSource> dataSourceList = cgDataSourceService.findBySearch(new CgDataSource());
			request.setAttribute("dataSourceList", dataSourceList);
		} catch (Exception e) {
			logger.error(e.getMessage(), e);
		}

		return "generator/table/buildConfig";
	}

	@RequestMapping("/getTables")
	@ResponseBody
	public String getTables(Long dataSourceId){
		JSONObject json = new JSONObject();

		try {

			CgDataSource dataSource = cgDataSourceService.getById(dataSourceId);
			List<String> tables = new ReadTableUtils().readAllTableNames(dataSource);
			json.put("result", tables);
		} catch (Exception e) {
			json.put("result", "error");
			logger.error(e.getMessage(), e);
		}

		return json.toString();
	}

	@RequestMapping("/tableConfig")
	public String tableConfig(Long id,HttpServletRequest request){

		try {

			CgTableConfig tableConfig = cgTableConfigService.getById(id);
			request.setAttribute("tableConfig", tableConfig);
		} catch (Exception e) {
			logger.error(e.getMessage(), e);
		}

		return "generator/table/tableConfig";
	}

	@RequestMapping("/update")
	@ResponseBody
	public String update(CgTableConfig tableConfig){
		JSONObject json = new JSONObject();

		try {

			tableConfig.setUpdateUser(getCurrentUser().getId());
			tableConfig.setUpdateDate(new Date());
			cgTableConfigService.update(tableConfig);
			json.put("result", "save_success");
		} catch (Exception e) {
			json.put("result", "save_fail");
			logger.error(e.getMessage(), e);
		}

		return json.toString();
	}

	@RequestMapping("/delete")
	@ResponseBody
	public String delete(Long id){
		JSONObject json = new JSONObject();

		try {

			cgTableConfigService.delete(id);
			json.put("result", "delete_success");
		} catch (Exception e) {
			json.put("result", "delete_fail");
			logger.error(e.getMessage(), e);
		}

		return json.toString();
	}
}

redpajama_set_name: RedPajamaGithub


Looking to buy or sell Motor vehicle chassis fitted with engine? List your company on Macro Market.
Motor vehicle chassis fitted with engine are the 717th most traded product and the 791st most complex product according to the Product Complexity Index (PCI).
The top exporters of Motor vehicle chassis fitted with engine are Sweden ($729M), Brazil ($609M), India ($392M), Japan ($343M) and Germany ($271M). The top importers are Iran ($268M), Nigeria ($182M), China ($180M), Bangladesh ($177M) and Spain ($174M).
Motor vehicle chassis fitted with engine are a 6 digit HS92 product.
This treemap shows the share of countries that export Motor vehicle chassis fitted with engine.
This treemap shows the share of countries that import Motor vehicle chassis fitted with engine.
This visualization shows products that are likely to be exported by countries that export Motor vehicle chassis fitted with engine.


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text:
News: UK Black Rock studio to close
In another blow to the UK industry, one that echoes the recent closure of Bizarre Creations, Disney confirmed yesterday that it would be closing down UK-based studio Black Rock after its most recent pitch failed to be greenlit. Although ostensibly the case, the move was certainly influenced by the failure of previous titles Pure and Split/Second to perform well commercially, a failure that led to a spate of voluntary redundancies two months ago at the studio.
Following the cancellation of sequels to Pure and Split/Second, the studio was directed to focus on Disney's current priority, social and free-to-play gaming. It's appears that the pitch that was rejected by Disney was based around these tenets.
Staff, speaking to Develop, cried foul, one source stating that ''Really, they were just paying lip-service to us with this talk of the pitch. Anything we suggested wasn't going to be accepted', another arguing that the studio 'never stood a chance'.
The parallels with Liverpool's recently closed Bizarre Creations are clear to see, both studios had enjoyed success previously and specialised in the racing genre. Similarly, both studio's recent games were well received critically but failed to perform as expected commercially. Perhaps crucially, both UK studios occupied the so-called middle-ground between triple-A titles and budget fare, an area increasingly squeezed by the growth of mobile and social gaming.
It's a sore loss to the UK industry to lose yet another talented studio, and here's hoping that the staff involved are successful in their future ventures.
[Source: Develop]
Posted by K S Rees at 13:57
Labels: blackrock, disney, game industry, news, uk
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The instrument used to gauge the amount of moisture in the soil is known as the soil moisture sensor. Accuracy and reliability are two main distinguishing characteristics of a great soil moisture sensor. So looking for a reliable soil moisture sensor will go a long way.
Your soil moisture sensor should be compatible with all delta-T devices loggers. Other data loggers should also be compatible with your soil moisture sensor.
You should measure the soil moisture at your garden with a top quality soil moisture sensor. The soil moisture sensor you buy should also be easy to use. You should buy a soil moisture sensor that is simple, reliable, accurate in readings and cheap.
The soil moisture sensor you buy should also offer data storage and download capability. The latest version of soil moisture sensor that is updated with the new technologies will be great.
You should also factor in the ease of installation. You should consider the ease of use of your soil moisture sensor. Being able to withstand high temperatures and salinity is a great characteristic of a great soil moisture sensor.
The level of water in the soil should be detected by a good soil moisture sensor. The level of saturation in the soil should be measured with a reliable soil moisture sensor.
You should buy a soil moisture sensor that is protected from destruction from moist conditions. There are high-quality soil moisture sensors that do not degenerate easily under the moist conditions so you should look for them when making purchase of a soil moisture sensor.
It is also possible to acquire an automated soil moisture sensor. Your automated soil moisture sensor could give you the readings that it took while you were not around.
It should be easy and enjoyable to take readings of the soil moisture sensor. A top quality soil moisture sensor will save the day.
While buying a soil moisture sensor you should consider factors such as technology, application, and research. An up to date soil moisture sensor would be great.
The soil moisture sensor helps a farmer know the right amount of water content in the soil. You should know that the use of soil moisture sensor should be a factor to consider. The information will enable you to make sound decisions when it comes to soil moisture sensors.


text:
Libyans Fight Against Last Qaddafi Holdouts
Oct. 18, 201: Revolutionary fighters fire at Qaddafi loyalists in downtown Sirte, Libya. (AP2011)
SIRTE, Libya -- Libyan revolutionary forces fought building by building Wednesday against the final pocket of resistance in Muammar Qaddafi's hometown -- the last major city in Libya to have been under the control of forces loyal to the fugitive leader.
While Libya's transitional leadership worked to consolidate control over the entire country, the country's acting prime minister warned in a newspaper interview that Qaddafi can still cause trouble from his hiding place.
Mahmoud Jibril was quoted by the pan-Arab Asharq Al-Awsat paper Tuesday as saying that the ousted leader is moving between Niger, Algeria and the vast southern Libyan desert and has been trying to recruit fighters from Sudan to help him establish a separate state in the south, or to march to the north and destabilize the new regime.
The report could not be confirmed, but it underscored fears that the inability to catch Qaddafi, who escaped with two of his sons after revolutionary forces swept into Tripoli in late August, would allow him and his supporters to wage an insurgency.
"Qaddafi has two options: either to destabilize any new regime in Libya or to declare a separate state in the south," Jibril was quoted as saying, adding there was evidence about this but he didn't elaborate.
Suggesting that the U.S. also was concerned about the possibility, Secretary of State Hillary Rodham Clinton said during a visit to Tripoli Tuesday that she hoped Qaddafi would be captured or killed.
Although two months have passed since Qaddafi fled the capital, Libya's new leaders have refrained from declaring national "liberation" until the fall of Sirte, which Qaddafi transformed from a fishing village into a modern city after he seized power in 1969.
Revolutionary forces on Tuesday pushed from the east into the small pocket of the city under the control of Qaddafi loyalists and captured a vegetable market, though they came under heavy fire from snipers and rocket-propelled grenades on the rooftops of residential buildings and homes along major streets.
On Wednesday, Wissam bin Hmade, the commander of one of the revolutionary brigades from the eastern city of Benghazi, said they had the Qaddafi supporters corralled in a 700 square meter residential area but were still facing heavy rocket and gunfire from snipers holed up in surrounding buildings.
It took the anti-Qaddafi fighters, who also faced disorganization in their own ranks, two days to capture a single residential building.
Another commander, Khaled al-Maghrabi, said 15 fighters were killed in a friendly fire incident.
It is unclear whether loyalists who slipped out of the besieged cities of Bani Walid, which was captured this week, and Sirte might continue the fight and attempt to organize an insurgency using the vast amount of weapons Qaddafi was believed to have stored in hideouts in the remote southern desert.
Unlike Iraq's Saddam Hussein, Qaddafi had no well-organized political party that could form the basis of an insurgent leadership. However, regional and ethnic differences have already appeared among the ranks of the revolutionaries, possibly laying the foundation for civil strife.
Qaddafi has issued several audio recordings trying to rally supporters. Libyan officials have said they believe he's hiding somewhere in the vast southwestern desert near the borders with Niger and Algeria.
The whereabouts of two of his sons also remain unknown, although commanders have said they believe Muatassim and Seif al-Islam are hiding in Sirte and Bani Walid, respectively. Seif al-Islam had been Qaddafi's likely choice to succeed him as Libya's leader.
Anti-Qaddafi fighters combed Bani Walid on Tuesday for signs of Seif al-Islam and other high-level regime figures in the desert enclave, 90 miles southeast of Tripoli.
"Seif was seen on Thursday. He was eating in a desert village close to the city," one field commander, Said Younis, said.
The Netherlands-based International Criminal Court has charged Seif al-Islam, his father and Qaddafi's former intelligence chief Abdullah al-Senoussi with crimes against humanity for a brutal crackdown on the uprising.


text:
Local Limelight
Financial Buzz
August 2017 Around Town
Now Open – Del Mar's First Brewery, Viewpoint Brewing Co.
On Wednesday, July 19, Viewpoint Brewing Co., Del Mar's first craft brewery, held its grand opening, coinciding with the Del Mar Thoroughbred Club's Opening Day. Founded by local chef and home brewer Charles Koll, the brewery and restaurant is located next to the Del Mar Fairgrounds just off the 5 freeway's Via de la Valle exit. Its arrival brings to Del Mar a seasonal restaurant focused on changing the point of view of traditional gastro pub dining alongside its namesake craft beer. ViewpointBrewing.com
SDRVC Receives Grants for River Path Del Mar Extension
The San Dieguito River Valley Conservancy (SDRVC) has been awarded two grants to fund the studies needed to move forward with the design and planning of the River Path Del Mar Phase 3 Extension Project, the final segment of the path. REI and The Malk Nature Fund each have granted the SDRVC $15,000 to cover the estimated $30,000 cost of completing the topographical and right-of-way mapping and biological studies. SDRVC is working closely with the City of Del Mar on the River Path Del Mar project. SDRVC.org
Nativa Interiors Remodels Solana Beach Location
Local design firm Nativa Interiors fully remodeled their Solana Beach location recently. All architectural structures and cabinetry was built in their factory, the same factory where they build the furniture they sell at their stores. John Hayward, one of the senior designers at the company, designed the space. The redesign comes after nine years at the same location in Cedros. NativaFurniture.com
VALENTI INTERNATIONAL IS DMTC OPENING DAY TITLE SPONSOR
Thoroughbred race fans celebrated San Diego's biggest equestrian event in style this year, with Valenti International as title sponsor of Opening Day at Del Mar. More than 40,000 fans were in attendance on Wednesday, July 19, for one of the most fabulous, fashionable and fun festivities in the west. Valenti International featured signature touches throughout the track, including a winner's circle presentation and judging the "Most Glamorous" hat in Del Mar's highly successful Opening Day Hats Contest. Valenti International will also be partnering with the Del Mar Thoroughbred Club for another historical equestrian event, as title sponsor of the Breeders' Cup Barn at the Beach, October 28 – November 4, 2017. ValentiInternational.com
Local author pens sci-fi novel
Todd Wonkka, Assistant-Pro at the Del Mar Country Club for 8 years now, is soon releasing his first novel titled Under The Rug. A gripping story about friendship and finding one's self, set against the backdrop of a provocative Hollywood lifestyle, Wonkka describes it as psychological fiction with a touch of satire. The novel will be released on Inkshares. Show your support for Wonkka by pre-ordering your copy/copies today! Inkshares.com/books/under-the-rug | ToddWonkka.com
KAABOO Del Mar's HUMOR ME Comedy Lineup
KAABOO Del Mar has announced its 2017 HUMOR ME comedy lineup for its third annual live music and entertainment experience happening September 15-17, 2017. Take a break from dancing to sit back and laugh with nationally recognized comics at HUMOR ME, KAABOO's indoor comedy club. Late-night show regular Sebastian Maniscalco, actor and former Saturday Night Live comedian David Spade, and Grammy-winner for 2017's Best Comedy Album, Patton Oswalt, are among the nation's funniest names in comedy headlining this year's HUMOR ME stage. KaabooDelMar.com
Resounding Joy Moves To New Location
Resounding Joy and MusicWorx has moved to a new location since June, one mile down Sorrento Valley Rd. The new address is 10455 Sorrento Valley Rd., Ste 208 San Diego, CA 92121. They offer individual and group music experiences in the relocated Music Wellness Center, including a robust music therapy program for people with Parkinson's. ResoundingJoyInc.org
Jazz Under The Stars Presented By Smooth Jazz KIFM
Enjoy live smooth jazz at Pala Casino's Starlight Theatre on Saturday, August 12th. The show stars renowned singer/songwriter/musician Bobby Caldwell (What You Won't Do For Love), along with international jazz trio, The Foreign Affair Trio featuring Michael Lington, Alex Bugnon, and Marc Antoine. Tickets range from $15-$35 with VIP tickets at $65 and can be purchased online. Smooth Jazz KIFM is back in San Diego and can be found on 98.1 HD-2, streamed at KIFM.com or through the Sunny 98.1 app. KIFM.com | StarTickets.com
Breeders' Cup Barn at the Beach Special Events
The Breeders' Cup "Barn at the Beach" will be the site for a week of events surrounding the much-anticipated Breeders' Cup World Championships coming to the Del Mar Thoroughbred Club. The Festival Week is set for October 28 – November 4. The Barn at the Beach will be a unique oceanfront venue providing a dramatic gathering place for attendees from across the globe and local community members to come together for special events celebrating the internationally acclaimed horseracing event Del Mar. VisitDelMarVillage.com/breederscup2017 | BreedersCup.com
Boys & Girls Clubs of San Dieguito's Horseshoe Corn Hole Tournament
Chris Roth and Brett Jerhoff the Jr. Board charity Horseshoe Corn Hole Tournament August 26th at Del Mar Dog Beach. These successful and charming young gentlemen, along with the rest of this well-established board, bring together food, craft beer, and hundreds of fun and sun loving professionals for this premier annual North County event. Like the Jr Board generally, it's a little networking, mixed with a lot of fun and just the right amount of chilled foam to top it off. BGCSanDieguito.org


text:
Q: One-dimensional family of complex algebraic K3 surfaces Let $X$ be an algebraic complex K3 surface, we know that $X$ is deformation equivalent to a smooth quartic surface or more generally a K3 surface with Picard number $1$ (a very general K3 surface in the family of K3 surfaces). It means that there exists a proper morphism $\mathcal{X}\rightarrow S$ with each fibre a K3 surface, $\mathcal{X}_{\eta}=X$ ($\eta$ a general point on $S$) and $\rho(\mathcal{X}_t)=1$ for some closed $t\in S$.

I want to show that $S$ can be a smooth algebraic curve or better be $\mathbb{A}^1$.

A: Choose any ample class in $\mathrm{Pic}(X)$, assume its degree is $d$. Let $M_d$ be the moduli space of polarized K3 surfaces of degree $d$ with appropriate level structure so that it has a universal family (alternatively one can use here the $\mathrm{Quot}$-scheme that is used to construct the moduli space). Let $x \in M_d$ be the point that corresponds to $X$. Let $S_0 \subset M_d$ be a general curve in $M_d$ through $x$, and let $S \to S_0$ be a resolution of singularities of $S_0$. Then the pullback to $S$ of the universal family from $M_d$ is a family with the required properties.

redpajama_set_name: RedPajamaStackExchange


text:
package com.tribbloids.spookystuff.uav.planning.VRPOptimizers;

import com.graphhopper.jsprit.core.util.VehicleRoutingTransportCostsMatrix;

import java.io.BufferedReader;
import java.io.File;
import java.io.FileReader;
import java.io.IOException;
import java.util.*;

public class MatrixReader {

//	public static void main(String[] args) throws IOException {
//		VehicleRoutingTransportCostsMatrix.Builder builder = VehicleRoutingTransportCostsMatrix.Builder.newInstance(true);
//		new MatrixReader(builder).read("/Users/schroeder/Documents/jsprit/abraham/Matrix.txt");
//		VehicleRoutingTransportCostsMatrix matrix = builder.build();
//
//		System.out.println(matrix.getTransportCost("18", "19", 0, null, null));
//	}

	private VehicleRoutingTransportCostsMatrix.Builder costMatrixBuilder;

	private Map<String,Double> distances = new HashMap<String,Double>();

	private double probRelation = 0.0;

	private double percentSpeedup = 0.0;

	private Random random = new Random(Long.MAX_VALUE);

	public MatrixReader(VehicleRoutingTransportCostsMatrix.Builder costMatrixBuilder) {
		super();
		this.costMatrixBuilder = costMatrixBuilder;
	}

	public MatrixReader(VehicleRoutingTransportCostsMatrix.Builder costMatrixBuilder, double probRelation, double percentSpeedup) {
		super();
		this.costMatrixBuilder = costMatrixBuilder;
		this.probRelation = probRelation;
		this.percentSpeedup = percentSpeedup;
	}

	public void setRandom(Random random){
		this.random = random;
	}

	public double getDistance(String from, String to){
		if(from.equals(to)) return 0.0;
		String key = from+"_"+to;
		if(!distances.containsKey(key)){
			key = to+"_"+from;
		}
		if(!distances.containsKey(key)) throw new IllegalStateException("key " + key + " does not exists");
		return distances.get(key);
	}

	public void read(String filename) throws IOException{
		BufferedReader reader = new BufferedReader(new FileReader(new File(filename)));
		String line = null;
		List<String[]> dataset = new ArrayList<String[]>();
//		List<Double> distances = new ArrayList<Double>();
//		List<Double> times = new ArrayList<Double>();
		while((line=reader.readLine())!=null){
			String cleanedLine = line.trim();
			String[] tokens = cleanedLine.split(";");
			if(tokens.length == 4){
				if(!cleanedLine.contains("Distance")){
					dataset.add(tokens);
					String distance = tokens[2].replace(",", ".");
					double dist = Double.parseDouble(distance);
					String time = tokens[3].replace(",", ".");
					double t = Double.parseDouble(time);
					costMatrixBuilder.addTransportDistance(tokens[0], tokens[1], dist);
					double randomNumber = random.nextDouble();
					boolean speedupRelation = randomNumber < probRelation;
					if(speedupRelation){
						costMatrixBuilder.addTransportTime(tokens[0], tokens[1], (t - t*percentSpeedup));
					}
					else{
						costMatrixBuilder.addTransportTime(tokens[0], tokens[1], t);
					}
					distances.put(tokens[0]+"_"+tokens[1], dist);
				}
			}
		}
//		Comparator<Double> comparator = new Comparator<Double>(){
//
//			@Override
//			public int compare(Double o1, Double o2) {
//				if(o1 < o2) return -1;
//				if(o1 > o2) return 1;
//				return 0;
//			}
//
//		};
//		Collections.sort(distances, comparator);
//		Collections.sort(times, comparator);
//
//		for(String[] data : dataset){
//			double dist = Double.parseDouble(data[2].replace(",", "."));
//			double time = Double.parseDouble(data[3].replace(",", "."));
//			System.out.println(data[2]+";"+data[3]+";"+distances.indexOf(dist)+";"+times.indexOf(time));
//		}
		reader.close();
	}

}


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