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- anotherbee
- 8 years ago
- Wedding: August 2011
…and nothing. No pos OPK, no pos HPT, no CM…nothing.
…and nothing. No pos OPK, no pos HPT, no CM…nothing.
@anotherbee: 🙁 Sorry to hear, this sounds super frustrating. Are you charting at all or just using OPK's? I hope something happens for you soon…. 🙁
I'm sorry! That is so frustrating. My longest cycle was 61 days. If I went longer the dr. was going to prescribe something to help me start my cycle. Have you called your doc?
Thanks ladies. I'm charting, and my temps are all over the place. I'm waiting to get into an OBGYN, but it likely won't be until June. His nurse (my aunt) tells me there is nothing my GP can do for me anyways, and I believe it since she told me charting wasn't accurate for confirming O.
I'm just sharing my misery 🙂
@anotherbee: I'm going to bite my tongue for the most part, because I'm pretty sure you know my opinion, but I will reiterate that I think you should be insisting that you see a doctor much sooner. Charting or not, it's not healthy to go that long without a period. I once went 6 mos. without mine — most of it because I didn't notice how long it had been, but also because it took me about a month to get in with the ob/gyn. When I finally was prescribed provera and took it to induce my period, it was the worst PMS of my life. I was miserable, depressed and irritated for days.
@anotherbee: I can' remember, are you recently off BCPs? If you normally have a "regular" cycle and aren't recently off BCPs there is definitely medication that can "kick start" your cycle. Most doctors will want your body to do what it should naturally, but after 72 days, I wouldn't think it was out of the question to at least call your doctor to get some information over the phone.
Is it normal for your doctor to be unavailable for consultation for a month+ ?? I'd be looking for a new doctor, personally, if that was the case!
@bunny: haha, I do know your opinion 🙂
everyone else:
my GP is new to me, and I dont' like her, but GPs are hard to come by, and I'm lucky to have her. I can get in to see her with no trouble (usually within 3 days), but she's not specialized.
I'm waiting to get into the OBGYN. He's one of the best in the city, and as a result, very popular and busy. My aunt is his RN, so she's getting me in as soon as it's possible, unfortunately, he has alot of "urgent" cases right now. So that's why I'm waiting so long. Typically, it would be MUCH longer. I'm hesitant to ask for a different dr because I know he's good, and knows my family history (all my cousins have gone to him/our aunt).
I've been off HBC since Aug, and was averaging 40 day cycles. My longest was 50 some days, when I o'd on CD 37. This is only cycle 5 since coming off HBC 8 months ago.
I asked my aunt if I should wait it out or go see my GP, and she suggested I wait it out, since the GP won't be able to do anything for me (I think she lacks and understanding of assisted ovulation). When I went to see her originally, her first statement was "those drugs for fertility are really strong". So she skipped over the whole "lets find out WHY" part…
I'm 29, 30 in Oct, and have been on HBC since 17ish. never had regular cycles (I remember skipping months, but never tracked), but only started my period around 16 anyways, so not alot of time to regulate.
I was on BCPs for about as long as you then. My cycles were regular when I first started at 13 but then I started track and didn't have one for 5 months. Then they were painful and irregular so I went on BCPs. When I went off of mine, I had a period about 5 days later and nearly passed out at work twice. Had to go to the ER, my GYN thought I was miscarrying. She found out I wasn't…it was just the buildup of hormones. But I've been regular ever since.
I think (but I'm no doctor) that your cycles seem pretty long and not in the "normal" range. It could be hormonal. I still think I'd go see the GP and get her to assist you with ending this cycle. I personally don't think I'd want to wait until June.
FWIW, after my 6 mos w/ out a period, followed by the period from hell, I was regular (even if my cycles were a little longer than average), for about 2 years until I went on BCP.
@anotherbee: I totally get not wanting to go see your GP that doesn't specialize in this sort of thing- I would agree that they probably aren't the doctor to see. In your post you put that typically it would take much LONGER to see your OB than it is taking right now? I can really understand wanting to see a qualified, well-known doctor as well. But personally, I would want a doctor I had more access to though- do you want to have to be an "urgent case" in order to have access to your doctor throughout a pregnancy? Just a thought. If you are not looking to change doctors, maybe you can ask your aunt if she thinks you could schedule a phone call with the doctor- perhaps if he can call you after hours, or on his way home, he'll be able to get in contact with you sooner.
EDIT- when I re-read my post, it sounds a little judgemental and I don't mean for it to sound like that. Please don't take it that way:) I think you should do whatever you're comfortable with- I was just expressing how I would feel:)
It wouldn't even have to be assisted ovulation meds. I mean it's pretty obvious that ovulation isn't occuring, but the focus right now should be on health. Not having AF is unhealthy after a couple of months. Your body needs to shed lining. Taking meds like provera or prometrium might not get you ovulating, but it WOULD at least keep your body healthy lining-wise. That was what my OB told me when she found out that it had been over 6 months since my last AF…
thanks for the advice guys, trust me, it's all gone through my head 🙂
@MRSLMA: not judgemental. I agree. The reason I'm having to wait so long is because I'm not his patient yet. I need to get "squeezed in" between his regular patients (who are all preggers). Once I'm actually on the books as a patient, then I'll be able to get into see him no probs.
As an example of how long of a wait specialists have, Darling Husband got a referal to a urologist back before Christmas. He got the letter in the mail last week, his apt is in July! Crazy! But, we're in canada and it's free, so I don't complain.
@anotherbee: Ok now I understand. I was in England when I had a cyst burst and the hospital wanted to admit me for the weekend b/c the U/S tech wasn't coming in until Monday. I left and told them to schedule me an appt. It took me 4 months to get in for that and of course, it was gone. It's one of the reasons I wasn't too happy with free healthcare. I guess I would wait then until you can see him. Especially since you are wanting to be a regular patient of his.
@anotherbee: that makes SO much more sense…. and seems like a reasonable time frame! Even in the states, to become a patient of a highly recommended specialist would take months too.
I'm hoping you get some answers soon… I can't imagine how frustrating it is to have to wait and wonder what's going on inside! Hopefully the doctor will have some insight and a plan of action!
The topic 'CD 72….' is closed to new replies.


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Vera Farmiga knows that she's going to die on "Bates Motel." Everyone who's seen "Psycho" knows what will happen to Norman's poor momma. Farmiga discusses the series with Gold Derby editor Tom O'Neil.
Vera Farmiga should win an Emmy solely for this performance.
What better place to chat with the star of A&E's hit show "Bates Motel" than at the actual Bates Motel & Mansion itself? That's just what YH does as we head to the Universal Studios Hollywood backlot to catch up with actress Vera Farmiga to get all the "licentious" dish on Season 3 of the series based on the classic movie 'Psycho'! She gushes about her co-star Freddie Highmore, plus she talks about some strange requests and fan encounters and reveals the inspiration for her take on the enigmatic Norma Bates!
Golden Globes exclusively interviews Vera Farmiga on her show "Bates Motel"
In this scene from the second After Hours Special, Vera Farmiga, who plays Norma Bates, tells how she treats the cast to elaborate meals during holidays.
In this scene from the After Hours Special, Vera Farmiga explains Norma's unease over Norman's obsession with death, and how she scored lower than Norma on a parenting quiz.

subset: commoncrawl


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We are involved in many different kinds of investments in our lives. The main reason of doing investments is to earn some extra money in addition to our income. This money can help us in many different needs which motivate us to invest at places where we can have a fair amount of money returns. Also, it is helpful for keeping our money safe for a certain period of time until we had done investment.
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24 Options Review
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Angharad is a Clinical Geneticist with a subspecialist interest in cardiac conditions; and a postdoctoral research fellow in the Cardiovascular Genetics and Genomics Group. She is interested in the diagnosis of complex cases, delineating and understanding the variability in clinical expression of recognised syndromes (expanding the phenotype) and identifying new syndromes and diagnoses to inform both clinical practice and research.
We are also launching a community web portal - the Heart Hive - to support patient participation in research into heart muscle disease, allowing participants to self-enrol in our research, and to see what other research opportunities are available.
et al., 2019, A gene-centric strategy for identifying disease-causing rare variants in dilated cardiomyopathy, Genetics in Medicine, Vol:21, ISSN:1098-3600, Pages:133-143
et al., 2017, Phenotype and Clinical Outcomes of Titin Cardiomyopathy, Journal of the American College of Cardiology, Vol:70, ISSN:0735-1097, Pages:2264-2274
et al., 2016, Titin truncating mutations: a rare cause of dilated cardiomyopathy in the young, Progress in Pediatric Cardiology, Vol:40, ISSN:1058-9813, Pages:41-45
et al., 2015, Integrated allelic, transcriptional, and phenomic dissection of the cardiac effects of titin truncations in health and disease., Sci Transl Med, Vol:7
et al., 2013, Next Generation Diagnostics in Inherited Arrhythmia Syndromes, Journal of Cardiovascular Translational Research, Vol:6, ISSN:1937-5387, Pages:94-103


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Product Description
What could be better than a magic dress shop, where each gorgeous outfit takes you on an adventure through time and imagination . . .?!
In this second book in the series, Ava puts on a beautiful twinkling tutu and finds herself magically whisked away to Victorian times – where she has an important part to play in an extravagant ballet show . . . and making a girl's dreams come true!
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26 Apr - min - Uploaded by Radio 1 Essential Mix 1) Kryder & Tom Staar - Jericho 2) Kryder - K2 3) Kryder - Aphrodite 4) Renato Cohen. 21 Mar - min - Uploaded by DancePolice http://www. youtube. Kryder brings the noise to the Essential Mix. (Jewel Kid Rework) vs Funk A Tron vs Witch Doctor (Kryder Edit) ID vs Pyramid (Kryder Edit).
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Cabinets One is a specialty retailer in the hard surface flooring market, offering the broadest in-stock selection of tile, wood, and laminate flooring at everyday low prices.
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Phone: +372 66 44 055
Kolm Sibulat – onions that are not bitter
Kolm Sibulat is a paradise for a food enthusiast. Their dishes are simple, but always very delicious. And their drinks' menu is increasingly more interesting. All of this for a price that soon allows you to visit the restaurant again. So, these onions are pretty sweet?
But true onions must have a bit of bitterness. Kolm Sibulat is bitter to their competitors, who often do not understand how a simple eatery can be on a higher position in this chart than many restaurants that are finer and have more ambitions.
The most difficult thing is to prepare the simplest dishes well. Big ambitions and high self-esteem are not good flavourings; often they can ruin the food instead. Less bitterness – that seems to be the message of Kolm Sibulat.
Telliskivi 2, Tallinn


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What marketplace business model is right for you? Here, you learn how to choose the best business model for your business.
What marketplace business model should you choose? After all, picking the wrong business model is one of the main reasons why businesses fail. If you want to learn exactly how to choose the best business model for your marketplace, read on.
Source: Pexels.com
Why is marketplace as a business model so attractive?
First: What makes a peer-to-peer, B2B, or B2C marketplace such an attractive business model?
Good question. The thing is: You want to make sure you're working on the most lucrative business possible. And that's exactly what a marketplace is.
What is a marketplace?
An online marketplace is a platform that facilitates transactions (monetary or based in another form of value) between users. The owner of a marketplace doesn't own inventory. Instead, a marketplace owner provides the means for people to find each other and complete a transaction.
There are different types of marketplaces. You have peer to peer marketplaces, which enable transactions more generally between people (think, Airbnb as long as the renter is an individual and not a rental company). Then there are business-to-consumer marketplaces where consumers can find different businesses (like Amazon) and business to business marketplaces where businesses find each other (for example freelance marketplaces like Upwork).
Marketplace business model definition
In his talk on marketplaces, a16z general partner Jeff Jordan lists the reasons why marketplaces are superior to classic ecommerce and retail: identity, reputation, payments, trust and safety, and transparency.
When a marketplace achieves a level of growth, its value increases manyfold. This is because of the network effect, which means that the entire network is more valuable when more people use it because users benefit from each other. It's much harder to compete with a business that has built a network rather than a traditional ecommerce supplier, which makes the marketplace position itself sort of like a monopoly in a niche or industry.
We've talked about finding your marketplace business model with a marketplace business model canvas. But what about your financial model? That's what we'll look at next.
The best financial model for marketplaces
How do you decide which financial model to pick for your marketplace? Your financial model must be scalable, while at the same time meeting customer demand. If you prioritize one over the other, your marketplace will likely fail. So how do you balance all of this? Let's find out.
The first pricing model for marketplaces is a commission-based model.
Here, your marketplace makes money every time a transaction is made. You often get a certain percentage or amount based on the amount that is transferred. Commissions are usually the most lucrative revenue stream for marketplaces, especially as they grow and more and more transactions take place.
How much commission to charge
But how do you know how much to charge as a commission? And should you go for a percentage or a fixed commission? What you need to keep in mind is this:
- Your competition. Look at your competition. What commission do they take? This doesn't necessarily determine your entire pricing structure, but it can give a good indication of what works in your niche and industry.
- Marginal cost. How much does it cost to produce one additional unit of a product or service? This does affect your commissions because if they are too high, the profit margin for sellers will simply be too low for them to have an incentive to continue using your marketplace platform. For example, Etsy, where people sell handmade and vintage items with a higher marginal cost, takes 5% as a transaction fee. Upwork, a freelance platform, charges between 10-20%.
- What works for your users. Finally, tweak your commission model if necessary. In the end, your commission depends on what works for your users.
How to fix platform leakage
One roadblock for marketplaces that use commission-based pricing is platform leakage. This means that users go around the platform to deal with each other directly, without your platform as the middle man.
Platform leakage happens when users don't see enough value in your platform and perceive your commission as too high. For example, a freelancer on a freelance platform might contact a client directly outside of the platform if the commission is too high in comparison to the perceived value.
Fortunately, you as the marketplace owner can fix this. How? By giving the type of value that will make using your platform a no-brainer. Usually, this comes down to security and/or comfort.
Take home sitting marketplace TrustedHousesitters or house rental marketplace Airbnb. Both marketplaces have a need to make house owners feel secure on their platforms. That's why TrustedHousesitters offers a 24/7 veterinary advice line so homeowners can be sure their pets are in safe hands, as well as insurance for damage to property. Airbnb offers insurance for up to $1 million, which includes third party claims.
Alternatives to commissions as a pricing model
Note that commission as a pricing model doesn't work for all types of marketplaces. If all transactions on your marketplace are free (think: a marketplace built on service exchanges like TrustedHousesitters), a commission won't make much sense. In that case, another type of pricing model works best.
Plus, commissions are usually used together with another pricing model. For example, XXX.
Membership or subscription fee
If you use the membership or subscription fee pricing model, all or some of your users pay a monthly or yearly fee for using your platform. This model is perfect for platforms that provide high value (e.g. helps businesses find new clients), but where it's hard to charge a commission.
Now, every marketplace has the "chicken and egg problem" to some extent, so how to find sellers without customers and vice versa. But with a membership pricing model, you'll experience this even more. Without sellers or buyers, there simply isn't enough value for people to be incentivized to pay the membership fee.
How do you solve this? When you first set up your marketplace, you can either offer a discount to your membership fee or completely waive the fee for people who sign up.
For example, TrustedHousesitters charges an annual membership fee. The incentive is that people can get access to house sitters or houses that need house sitting without limits.
Listing fee
With a listing fee, people pay for listing their products or services on a platform. Listing fees are most commonly used when listings are valuable for the seller or service provider.
For example, Craigslist has built its pricing model around listing fees. And a lot of marketplaces tend to use a mix of listing fees and commissions. For example, Etsy charges both a commission (5%) and a listing fee ($0.20).
The problem with using only listing fees is that you don't get paid for the value of an item, but your listing fee can only be so high. You ultimately need a lot of listings to get enough revenue.
Lead fee
Lead fee means that you ask for a commission for specific leads (rather than when a transaction is completed). For example, this could be by having leads come to your site and sellers pay in order to bid to these leads. Most commonly, B2B marketplace platforms, and potentially B2C service platforms, use this pricing model. One of the pricing models Upwork uses is a lead fee for freelancers who bid for a job.
If your marketplace is a place where people share products or services for free, you'll need to be a bit more creative in how you monetize your platform. One way is to offer premium services. For example, a premium account might give people access to things like insurance, customer support, and other perks.
Your premium services need to be tempting enough for a portion of your users to be sustainable and scalable. Often, offering exclusively premium services won't be scalable enough as you need staff to manage premium services. That's why you might need to combine a freemium model with another pricing model, such as featured listings.
Featured listings and ads
With featured listings and ads, you make it possible for sellers to buy more visibility for their listing. For example, their listings might get higher up in searches or appear as the first search listing. Sites like Yelp use this pricing model.
Usually, featured listings and ads are used when it's free to use a site, but to stand out, users need to pay. In order to make this model sustainable, you need a good amount of users.
Want to get started building a profitable marketplace business today?
There you have it. Now you know which marketplace business models there are and which one to choose for a profitable marketplace business. In the end, commission together with another pricing model works great for most types of marketplaces.
To build a successful marketplace, you also need a powerful and easy to use marketplace platform. And to help you find the best platform, we made it free for you to try Kreezalid for 14 days. Click here to sign up!


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Cross the snow valley is a Parent-Child interactive theme amusement equipment. Tourists can take the Snow train and start a fantastic journey by interacting with lovely animals in the snow valley. Tourist can experience a fairy-tale dreamy journey by reality sense. We could also customize IP in the train according to investors' requests. Now it is the top choice for FEC.
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[[Hi Sister Laurel! Recently you put up the horarium (schedule) of your days on retreat, but I was wondering what your horarium for your days is usually like. It is not included in the copy of the Rule of Life you put up last year. Would you mind sharing that? Also, if someone wants to create a schedule for themselves, what should they pay attention to? Thank you!]]
I think if someone is trying to work out a schedule for themselves they have to strive to embody balance and order. As you will see, my own schedule (horarium) is oriented around liturgical prayer, lectio divina, study/writing, and quiet prayer (which actually accompanies all the other activities either before or after them). Those are the main elements, the things without which I don't function well and/or to which I am publicly committed to be faithful; this includes related practices or activities which fit around and support them.They are meant to alternate work, prayer, study, lectio, and I try to do this even when I cannot keep to a clock schedule. I also was given some great advice by the hermit monk who first read my Rule for suggestions. He said, "I hope you are careful to build in enough time for rest and recreation." Because of that, I take those things more seriously than I have at other times in my life, and everything is better because of it. I would certainly suggest people seeking to establish their own horaria do something similar.
In any case, here is the general weekday horarium for Stillsong Hermitage.
4:00 rising
4:15 Vigils.
4:30-5:30 quiet (contemplative) prayer
5:30 Lauds
6:00- 8:00 writing
7:00 breakfast (may skip on Fridays. may have after Mass)
8:30 Mass
9:15 Small chores around hermitage (during school year I first have coffee once a week on Fridays with parishioners at a small cafe run by school parents to benefit the parish "garden of learning"; during the Summer we go to coffee elsewhere.)
9:30 or 10:00 Lectio Divina: (Scripture)
12:30 Lunch (Dinner)
1:00 Rest, walk or nap
3:30 or 4:00 Writing, etc (work, theological study, occasional client). Afternoons are variable and may be taken up with whatever needs doing. This includes the period from 1:00 to 5:00pm.
5:00pm quiet prayer/meditation
6:00 Vespers (sung)
6:30 Supper and small chores
7:00 work (house chores, study, occasional clients --- depends on day. Wednesdays, orchestral rehearsal)
7:30- 8:30 Journaling (most days except Wednesdays and evenings with clients)
8:30 compline (sung)
9:00 pm bed --- earlier if no nap in afternoon (later on Wednesdays: @11:00pm)
Saturdays: as on weekdays until breakfast. Then, large chores, shopping, errands away from hermitage, etc.
5:00pm vigil Mass
6:30 supper
7:30 journaling, quiet prayer
9:00 compline
9:15 bed
Sundays: as on weekdays until breakfast. (May begin an hour later)
9:30am Mass at parish (time with friends afterward)
12:00 or 12:30 pm Lunch
2:00pm, quartets or quintets
4:15 quiet prayer (45 min-1 hr)
5:15 Vespers
6:00 supper and chores
8:00 journaling or lectio divina
9:30 Compline and bed
I tend to pick up email, check blog, etc several times a day, whenever there are a few extra minutes. On days when there are major errands to run I get less studying done, and a shorter time of lectio, but otherwise things go pretty much according to this schedule. During the Fall, Winter and early Spring, Wednesday evenings are set aside for orchestra rehearsals. Saturdays are the day for large errands, evening (vigil) Mass, etc. Sundays I often have quartets in the afternoon, and the whole day is a bit more relaxed; this is also the day I take Communion to neighbors unless they are seriously ill, in which case I will do that at any time, day or night. I may have lunch or dinner with friends (e.g., quartet members and their families), or just catch up on reading, etc, I have not had time for during the week.
Because I am active in my parish, and certainly more active than I was even a year ago (though this remains a limited thing), I do have to accommodate that in some way. Until recently I was simply trying to maintain regularity and balance in my daily horarium, but after this year's retreat it became clear that that is insufficient for me personally. Thus, besides the general daily horarium, each month I take a full week of strict anachoresis besides the more usual silence and solitude. My participation in any active ministry is the spillover of a contemplative life, not just one that includes contemplative prayer, so I am careful to be sure this remains true. What is generally the case is that every hermit determines how best to balance the solitary and the evangelical aspects of her vocation. Most do develop a rhythm like this, just as lay people and active religious may take a desert day every week or month. Sometimes a hermit may be accessible for several months and then retreat more completely for several. In any case, I do suggest that people trying to develop their own horaria experiment with desert days added either weekly or monthly. Hermits should experiment with extended periods of solitude to balance any periods where they are called on to minister in more active ways.
Hope this helps.
(The second picture is one of the clocks I have here in the hermitage. The other is a "zen clock" which allows me to set a timer for prayer or have the "alarm" (a single E flat bell) sound once an hour. (Yes, I respond to and order my life in some ways around bells, just as monastics have done for centuries!) I liked the contrast between the picture of the sundial and this particularly contemporary clock. Eremitical life has persisted in Christianity for almost two millennia. We live the tradition in a contemporary world.)
02 August 2008
Horarium Question
Posted by Sr. Laurel M. O'Neal, Er. Dio. at 1:03 PM
Labels: Catholic Hermits, daily schedule, developing a horarium, Diocesan Hermit, eremitical horarium, horaria


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Inward-facing conformation of glutamate transporters as revealed by their inverted-topology structural repeats
Edited by Christopher Miller, Brandeis University, Waltham, MA, and approved October 16, 2009
↵1T.J.C. and S.Q. contributed equally to this work. (received for review July 31, 2009)
Glutamate transporters regulate synaptic concentrations of this neurotransmitter by coupling its flux to that of sodium and other cations. Available crystal structures of an archeal homologue of these transporters, GltPh, resemble an extracellular-facing state, in which the bound substrate is occluded only by a small helical hairpin segment called HP2. However, a pathway to the cytoplasmic side of the membrane is not clearly apparent. We previously modeled an alternate state of a transporter from the neurotransmitter:sodium symporter family, which has an entirely different fold, solely on the presence of inverted-topology structural repeats. In GltPh, we identified two distinct sets of inverted-topology repeats and used these repeats to model an inward-facing conformation of the protein. To test this model, we introduced pairs of cysteines into the neuronal glutamate transporter EAAC1, at positions that are >27 Å apart in the crystal structures of GltPh, but ≈10 Å apart in the inward-facing model. Transport by these mutants was activated by pretreatment with the reducing agent dithithreitol. Subsequent treatment with the oxidizing agent copper(II)(1,10-phenantroline)3 abolished this activation. The inhibition of transport was potentiated under conditions thought to promote the inward-facing conformation of the transporter. By contrast, the inhibition was reduced in the presence of the nontransportable substrate analogue d,l-threo-β-benzyloxyaspartate, which favors the outward-facing conformation. Other conformation-sensitive accessibility measurements are also accommodated by our inward-facing model. These results suggest that the inclusion of inverted-topology repeats in transporters may provide a general solution to the requirement for two symmetry-related states in a single protein.
- alternating access
- conformationally sensitive cross-linking
- homology modeling
- neurotransmitter
- secondary transport
Glutamate is a ubiquitous signaling molecule in excitatory neuronal synapses. However, for efficient neurotransmission, and to avoid neurotoxicity, the concentration of glutamate in the synapse is carefully regulated through reuptake into the neuronal and glial cells surrounding the synapse. The proteins responsible for this uptake, the so-called excitatory amino acid transporters (EAATs), are capable of accumulating glutamate against a concentration gradient of up to 106 (1, 2). EAATs accomplish this by coupling glutamate transport to the passive diffusion of cations, first by cotransport of sodium and protons (1, 2), and subsequently by countertransport of potassium (3–5) (Fig. 1).
In recent years, crystallographic studies have revealed the atomic-resolution structures of several secondary transporter families. In the case of the EAATs, the structure of a bacterial aspartate transporter GltPh from Pyrococcus horokoshii (6, 7) has provided key insights into various aspects of transport, in strong agreement with biochemical, electrophysiological, and other experimental data (8). The GltPh structure contains eight transmembrane (TM) helices, as well as two helix-turn-helix motifs, HP1 and HP2, which point toward the center of the membrane from opposite sides. The substrate and ion binding sites are located close to the point where the tips of HP1 and HP2 meet. The available structures of GltPh, which vary in substrate or inhibitor occupancy, all correspond to a conformation resembling an outward-facing state, where the substrate binding site is essentially exposed to the extracellular solution (6, 7). Specifically, the substrate can become occluded by the closure of HP2, like a flap, whereas the pathway to the cytoplasm is blocked by ≈20 Å of packed protein. However, transport is generally believed to involve a mechanism in which the central binding site for substrate becomes alternately accessible to one side or the other of the membrane during the transport cycle (9). Here, we attempt to model the alternate, inward-facing form of the transporter using the presence of internal inverted-topology repeats in the structure.
We previously generated an atomistic model of an inward-facing conformation of an NSS transporter, LeuT, which contains an inverted-topology repeat of five TM helices, by swapping the conformation of the repeats in the outward-facing x-ray structure. That is, threading the sequences of the first five TM helices onto the structure of the second five TM helices, and vice versa, revealed a model in which the central binding site was accessible to the cytoplasm and closed to the outside. The change in conformation occurs because the five TM repeats in LeuT, although clearly related, are not identical in structure (10). Moreover, the small differences between them appear to be responsible for the structure adopting an extracellular-facing conformation in the x-ray structure. The cytoplasmic pathway revealed in the swapped model was in very good agreement with cysteine-scanning mutagenesis and accessibility measurements, as well as with the pathway of a related protein, vSGLT, for which a structure was subsequently reported in an inward-facing orientation (11). Consequently, we have proposed that such inverted-topology repeats might be an integral feature of transporters (10).
In the present study, we show that inverted repeats in GltPh structures can be used to model an alternate conformation of EAATs in which the substrate binding site is exposed to the cytoplasm. The large conformational change predicted by this model is supported by conformation-dependent cross-linking of site-directed cysteine mutants of a mammalian glutamate transporter, EAAC1. The model is also in harmony with conformation-dependent accessibility of engineered cysteines to the membrane-permeant sulfhydryl reagent N-ethyl maleimide (NEM) (12).
Structure of GltPh Contains Inverted-Topology Structural Repeats.
Previous analysis of the structure of GltPh suggested a structural relationship between the two hairpin loops and half of their adjacent helices (6). This relationship can be extended to include the entirety of TM7 and TM8, albeit with reduced structural similarity [supporting information (SI) Table S1 and Fig. 2A; see also SI Materials and Methods]. The resultant structural alignment provides better coverage of the protein structure, which is important for the modeling. These segments have opposite topologies in the membrane, and therefore one of them has to be rotated by ≈180° around an axis in the plane of the membrane to achieve the fit.
We also investigated the six TM helices in the N-terminal half of GltPh, which comprise the so-called "cylinder." This region has recently been suggested to contain a repeat of two TM helices (13). By including helix 4c (which spans the membrane), but excluding helices 4a and 4b (which do not span the membrane), we obtained a reasonable structural alignment between two 3-TM domains in this N-terminal cylinder (see Table S1 and Fig. 2B). The decision to exclude helices 4a and 4b (and the 3–4 loop) from the repeat is consistent with the large sequence insertion in this region in human EAAT homologues.
Based on the above analysis, the structure of GltPh can be described as containing four inverted-topology segments (which we refer to as I to IV) (Fig. 3A). That is, there is a pseudo 2-fold symmetry relationship between segments I and II, and another between segments III and IV.
In LeuT we observed a break in the symmetry between the five TM repeats, which was responsible for the formation of an asymmetric structure of the whole protein (10). In GltPh, by treating segments I and IV as one structural unit, and segments II and III as a second structural unit, and by superposing only segment I on segment II, we noticed a large difference in the position of the second half of each unit relative to the first half (Fig. 2C). Because the trimeric interface involving TMs 2, 4, and 5 is known to be unchanged during transport (14), we assume that any conformational changes within a given protomer would most likely involve segments III and IV rather than segments I and II. Consequently, it appears from Fig. 2C that the position of segment III is significantly lower within the membrane than the position of segment IV with respect to segments I or II.
Swapping the Conformations of Inverted Repeats Reveals a Cytoplasmic Facing Structure.
We used the presence of inverted-topology repeats in GltPh to model an alternate conformation of GltPh using the approach used for the NSS proteins, namely swapping of the conformations of the repeats (10). Because there are four repeat segments in GltPh rather than two, as in LeuT, we adjusted the protocol slightly. Thus, the conformation of segment I was modeled using the structure of repeat II as a template, and vice versa, while the conformation of segment III was modeled on IV, and vice versa (Figs. S1A and S2 and Fig. 3B).
The model of GltPh generated by swapping the conformations of the repeats contains a large conformational change with respect to the crystal structure, and appears to represent the cytoplasm-facing state of the protein (Fig. S1 C and D and Fig. 4A). Because the helices forming the subunit interface in the trimer (particularly TMs 4a, 4b, 4c, and 5) are likely to be fixed, according to evidence from cysteine cross-linking (14), we discuss the changes in the rest of the protein with respect to these helices. The major difference that can be observed is that the core of the protein containing the binding site is predicted to have moved toward the cytoplasm by 15 to 25 Å relative to the subunit interface (see Fig. 4 A and B). As a consequence, HP1 becomes exposed to the cytoplasmic solution, analogous to the extracellular exposure of HP2 in the x-ray structure (see Fig. S3 and Fig. 4A). This exposure of HP1 provides a means for release of substrate into the cytoplasm, namely by the flapping open of HP1, as has been suggested for HP2 in the extracellular conformation (7). In addition, the model predicts that HP2 will become buried under TMs 2 and 5 (see Fig. S3 and Fig. 4A), so that release of substrate and ions back to the extracellular environment would be inhibited significantly.
The model also suggests changes of 15 to 20 Å in the positions of TMs 7 and 8 to accompany the movements of HP1 and HP2 (see Fig. 4B). Such a concomitant movement may enable the binding site formed between these helices to be maintained. TMs 3 and 6, which shield TMs 7 and 8 from the lipid, are also shifted in the same direction, although to a lesser extent (10–15 Å). TM1 also moves somewhat, but in the opposite direction: that is, upwards in the membrane. In summary, the model predicts that the protein core consisting of HP1, TM7, HP2, and TM8 moves inward relative to the rest of the protein to form a cytoplasm-facing conformation that may correspond to an alternate state in the transport cycle.
Cysteine Cross-Linking Supports Close Approach of TM2 and HP2.
The large movements suggested by this model are supported by evidence for cysteine cross-linking in EAAT1 (15). Specifically, mutations to cysteine at positions equivalent to GltPh residues 52 or 55 (from TM2) and 364 (from HP2b) were found to cross-link in EAAT1, resulting in a loss of transporter activity (15). The Cα atoms of these residues are 27 to 29 Å apart in the x-ray structure of GltPh, and thus it is unlikely that they cross-link in this state (Fig. 5A). By contrast, in our model, TM2 has a significant interface with HP2. The distance between the Cα atoms in these cysteine pairs is ≈10 Å, which is significantly closer to the 7 to 8 Å expected for disulfide formation (see Fig. 5B). To test whether the cross-linking of HP2b and TM2 has a higher probability under conditions in which the transporter becomes inward-facing, we analyzed the equivalent double-cysteine mutants of EAAC1 (also known as EAAT3), with cysteines introduced at V420 of HP2, and at either R61 or K64 of TM2.
Aspartate transport by either R61C/V420C or K64C/V420C was stimulated by 50 to 70% upon addition of the reducing agent dithiothreitol (DTT) (Fig. 6 A and B), suggesting that the cysteine pairs spontaneously cross-link, similar to the observations for EAAT1 (15). Such cross-linking often inhibits transport (16–18), presumably because the disulfide imposes restrictions on the protein conformational changes necessary for transport, although such inhibition may also be the result of steric hindrance or another distortion introduced by the cross-link. Preincubation with 30 or 100 μM of the oxidizing agent copper (II)(1,10-phenantroline)3 (CuPh) resulted only in modest inhibition of transport, supportive of the notion that many of the cysteine pairs are already cross-linked. An almost full inhibition of transport was observed when the CuPh concentration was increased to 300 μM (see Fig. 6 A and B). Upon prior activation by DTT, the activity of the newly reduced transporters was fully sensitive to CuPh (see Fig. 6 A and B).
These results indicate that when cross-linking is complete, all transporters are locked permanently in one conformation so that the translocation cycle is blocked. Thus, when transport is measured subsequent to exposure to lower concentrations of CuPh (such as 100 μM used in the experiment depicted in Fig. 7), the remaining activity is a read-out of the proportion of those transporters that were not yet cross-linked.
The effects of DTT and CuPh on transport were only observed for the double-cysteine mutants, and not for the equivalent single-cysteine mutants (V420C, R61C, or K64C) expressed either alone or together: for example, V420C with R61C or with K64C (Fig. 6C). These results suggest that both cysteines need to be present on the same polypeptide chain for cross-linking to occur. Moreover, no effect of CuPh or DTT was seen with single-cysteine mutants in which the other residue was converted to either Ser or Ala, namely R61S/V420C, K64S/V420C, R61C/V420A, or K64C/V420A (see Fig. 6C). Thus, it seems unlikely that inhibition occurs as a result of cross-linking of one of the introduced cysteines to an endogenous cysteine.
Cysteine Cross-Linking Can Be Modulated by Substrate and Inhibitors.
To determine the effect of the conformation of the transporter on the rate of cross-linking, the initial activation of the double-cysteine mutants by DTT was followed by incubation for 5 min with 100-μM CuPh under various conditions. Transport was subsequently measured after washing out the CuPh. When the sodium-containing medium was either supplemented with glutamate or replaced by potassium, conditions that promote the formation of the inward-facing conformation (see Fig. 1), the inhibition by 100 μM CuPh was potentiated (see Fig. 7). This suggests that the cysteine residues are indeed closer together in the inward-facing conformation. By contrast, addition of the nontransportable substrate analogue D,L-threo-β-benzyloxyaspartate (TBOA) reduced the cross-linking effect of CuPh (see Fig. 7), consistent with the expectation that TBOA increases the proportion of protein trapped in an outward-facing conformation in which the cysteines are far apart. In the absence of external sodium (replacement with choline) there was very little impact, if any, of glutamate or TBOA (Fig. S4), apparently because glutamate translocation and TBOA binding both require sodium (see Fig. 1).
In principle, the modulation of the inhibition by CuPh could be a result of changes in accessibility of the engineered cysteine residues, rather than in their distance. As a measure of their aqueous accessibility, we determined the effect of MTS reagents on transport by the single cysteine mutants, although it is true that these reagents are larger than the reactive oxygen species generated by CuPh. Preincubation of R61C or K64C with MTSES (2-sulphonatoethyl-methanethiosulfonate) resulted in inhibition of transport, which was potentiated by either glutamate or external potassium (Fig. S5). However, TBOA, which protected against cross-linking of the cysteine pairs (see Fig. 7), did not modulate the inhibition of R61C or K64C by MTSES (see Fig. S5). For V420C, inhibition of transport by MTSET [(2-trimethylammonium) methanethiosulfonate] was protected not only by TBOA but also by glutamate (see Fig. S5), which again is different from the cross-linking results. Thus, while the accessibility of the introduced cysteines to MTS reagents appears to be dependent on the conformational state of the transporter, the effects of substrates and substrate analogues on cross-linking cannot be explained merely in terms of such changes in accessibility.
Experimental Evidence in Support of the Cytoplasm-Facing Model.
Available x-ray crystal structures of GltPh bound to either the substrate aspartate, or to a nontransportable analogue TBOA, have similar conformations (6, 7). Both are essentially outward-facing, although in the absence of TBOA the helix-turn-helix segment HP2 provides a thin barrier that appears to flap open to expose the substrate binding site to the extracellular solution (see Fig. 4A) (13). To date, proposed mechanisms of the conformational change required to expose this aspartate binding site to the cytoplasmic solution have invoked relatively small movements in the protein, involving mainly HP1, with the rest of the structure remaining essentially unchanged (6, 7, 19). Our atomistic model suggests that the cytoplasm-facing conformation of GltPh is a symmetry-related version of the extracellular-facing structures, and that the inward- and outward-facing states interchange through a large, concerted movement of the core binding region of HP1, TM7, HP2, and TM8 (see Figs. 4 and 5).
The prediction of a large conformational change in the EAATs is supported by the observation that HP2 and TM2 can spontaneously cross-link in both EAAC1 (see Fig. 6) and EAAT1 (15), even though the equivalent residues are >27 Å apart in the x-ray structures (see Fig. 5A), much too far to allow formation of a disulfide bond. Our model predicts a marked reduction of this distance to ≈10 Å. Furthermore, the fact that oxidative cross-linking is potentiated under conditions with increased proportions of inward-facing transporters, and reduced when the fraction of outward-facing transporters is increased (see Figs. 1 and 7), supports the idea that HP2 and TM2 indeed become closer in the cytoplasm-facing conformation of the transporter.
Large conformational changes are also supported by kinetic analysis of EAAC1, which showed that glutamate translocation is associated with energetic barriers of ≈100 kJ/mol (20). By contrast, distance measurements in EAAT3 using FRET suggest very little structural change in the protein (21). However, if the inward-facing states are relatively short-lived, then the latter measurements may not report on them, because FRET measures an average signal over all states.
Our model also predicts significant changes in solvent accessibility, so that most of HP1 and the cytoplasmic halves of TM7 and TM8 are accessible to intracellular solution in the model (Fig. S6 B and E), whereas they are buried within TMs 2, 3, 5, and 6 in the x-ray structure (Fig. S6 A and D). This prediction is consistent with the reactivity to NEM of cysteine residues introduced into these regions of a related transporter GLT-1/EAAT2 (12); this reactivity increases in the presence of extracellular potassium. We were able to match calculated and measured accessibility of many, but not all, of the residues in this region for the model based on Asp-bound GltPh (see Fig. S6 B and E). Closer agreement, particularly for residues in HP1, was observed for a second model in which HP1 is more open (Fig. S6 C and F). Disagreement between measured and calculated accessibility at a few positions may reflect inaccuracies in the models, such as slight rotational or translational errors in helix position, which can affect the calculated accessibility of individual residues. Differences between GLT-1 and GltPh may also affect the comparison. Nevertheless, the overall trend in these data (i.e., that these segments become more accessible in inward-facing conformations) is very consistent with the proposed model (see Fig. S6).
Analogous cysteine-accessibility measurements for HP2 of GLT-1 revealed that substrates and inhibitors have complex effects on their reactivity to the cysteine modifier MTSET (22). Some residues, such as 448 and 449 (GLT-1 numbering), are protected from reacting with MTSET in the presence of glutamate, consistent with being buried under TM2 in the inward-facing model (see Fig. 4A), as well as with a likely reduction in mobility of HP2. However, several of the reactive residues in HP2 of GLT-1 are also protected by the nontransportable substrate analogue kainate (22), even though kainate likely holds the protein in an outward-facing conformation. This apparent contradiction can be explained by an increase in contacts between HP2 and 3L4 (the loop between TMs 3 and 4) observed upon TBOA binding, which reduces the accessibility of residues in HP2 (7). Other residues in HP2 are, surprisingly, more accessible in the presence of TBOA than of kainate (22). Because some of these residues are on the underside of HP2, their accessibility may depend on the different degrees to which HP2 is wedged open by the two inhibitors.
Thus, the cysteine cross-linking, accessibility and other experimental data, are all consistent with the model of the inward-facing transporter. Clearly, however, ultimate proof of this prediction will require a crystal structure of a glutamate transporter trapped in this state.
A Mechanism of Alternating Access in EAATs.
Based on our data, we propose a mechanism of alternating access in EAATs involving four major states of the transporter: two outward- and two inward-facing (Fig. S7). The first state is a substrate-free, outward-facing conformation (or ensemble thereof) in which HP2 is open, similar to TBOA-bound GltPh (7), as observed in molecular dynamics simulations (23, 24). Upon ion and substrate binding, HP2 would then close in to form an outward-facing, occluded state (see Fig. 4A).
In the next step, the core segment containing HP1, TM7, HP2, and TM8 would undergo the movement shown in Fig. 4 to form the inward-facing conformation of the model. This conformational change probably requires close packing of HP1 and HP2 against TMs 7 and 8, so that together they form a smooth sausage-shaped bundle. Thus, in acting as wedges that hold HP2 open, substrate analogues, such as TBOA, may inhibit their own transport by preventing this major conformational change. In the resultant inward-facing, occluded conformation, the thin intracellular gate would be formed by HP1. Release of substrates would then require opening of HP1 to form an inward-facing, open state, which has been modeled here based on the TBOA-bound structure (see Fig. S6F). This four-state mechanism is consistent with trypsin cleavage of GLT-1, which suggests that glutamate is bound to two major conformations of the protein, only one of which can be inhibited by the nontransportable analogue dihydrokainate (25). Finally, binding of intracellular potassium would then favor closure of HP1, facilitating the reverse conformational change to the outward-facing state.
A remarkable feature of the inward-facing model of GltPh is that it was constructed only based on the structures of the inverted-topology repeats inherent in the EAAT fold. An essential step in the modeling, therefore, was the identification of structural symmetry within the GltPh structure to cover as much of the structure as possible. In GltPh, one can think of segments I and IV as forming one single, noncontiguous pseudorepeat, while segments II and III form a second pseudorepeat with inverted topology to the first (see Fig. 3). We observed an asymmetry in those two pseudorepeats, so that segment III is shifted vertically by ≈10 Å compared to segment IV (see Fig. 2C). This asymmetry reflects the location of the core protein segment (III and IV) within the cylinder (I and II), so that swapping the conformations of the repeats creates a vertical displacement of the whole III-IV segment, resulting in an inward-facing state.
Such observations not only provide a technique for predicting alternate conformations of transporters, but also give further support for the appealing idea of a role for inverted-topology repeats in the transport mechanism (10, 13). In such a mechanism, each repeat has the ability to adopt two distinct conformations with similar energies (10). Interchange between those conformations thus allows the formation of two different, but symmetry-related structures with analogous substrate pathways leading to opposite sides of the membrane, thereby elegantly solving the main requirements for alternating access.
Materials and Methods
Sequence Alignments.
An initial sequence alignment between the model and template was obtained from structure and ClustalW sequence alignments (26) of the four segments (see Fig. S1 and Fig. 3), and was adjusted to optimize, for example, binding site positions (see Fig. S2 and SI Materials and Methods).
Construction of the Protomer Model.
Models of GltPh protomers were built using Modeller 9v5 (27), where the template for each segment was the corresponding repeat from a GltPh crystal structure with PDB code 2NWL or 2NWW (see Fig. 3). Distance constraints were added to keep the internal conformations of certain local regions similar to the x-ray structure (see SI Materials and Methods). These distance constraints did not affect the overall conformational change predicted by the model.
Construction of Trimer Model.
To construct trimeric models, we fitted the protomers onto the x-ray structure using helices TM3, 4a, 4b, TM4c, TM5, and TM6 (residues 82–253), and minimized changes at the protein-lipid interface (see SI Materials and Methods).
Generation and Subcloning of Mutants.
The C-terminal his-tagged version of rabbit EAAC1 (28, 29) in the vector pBluescript SK− (Stratagene) was used as a parent for site-directed mutagenesis (30, 31). This was followed by subcloning of the mutants into his-tagged EAAC1, residing in the oocyte expression vector pOG1 (29) using unique restriction enzymes. The subcloned DNA fragments were sequenced between these unique restriction sites.
Expression of Transporters.
Complementary DNAs encoding the his-tagged EAAC1 and its derived mutants, subcloned in pOG1, were linearized with SacI, and cRNA was transcribed from each of the cDNA constructs with T7 polymerase and capped with 5′7-methylguanosine by use of the mMESSAGE mMACHINE (Ambion Inc.). Approximately 50 ng of the various cRNAs was injected into defolliculated stage V and VI Xenopus laevis oocytes and transport was assayed 3 to 4 days later.
Transport Measurements.
Uptake of d-[3H]aspartate was performed essentially as described (32), after pretreatment with DTT or CuPh, as detailed in the SI Materials and Methods.
We thank J. Faraldo-Gómez for valuable suggestions. This work was supported by SFB-807 from the Deutsche Forschungsgesellschaft (to L.R.F), the National Institute of Neurological Disorders and Stroke through National Institutes of Health Grant NS16708, and US–Israel Binational Science Foundation Grant 2007051 (to B.I.K.).
Note Added in Proof.
A 3.5 Å-resolution X-ray crystallographic structure of GltPh in which HP2 and TM2 are cross-linked by Hg2+ has recently been reported (34). This structure agrees remarkably well with our prediction of the major conformational change that occurs during transport, providing convincing evidence that inverted-topology structural repeats are responsible for the creation of two symmetry-related states in transporter proteins. Furthermore, our data for EAAC1 demonstrates that the predicted and observed conformational changes in the bacterial transporter also occur during transport of neuronal glutamate.
- 2To whom correspondence should be addressed. E-mail: lucy.forrest{at}biophys.mpg.de
Author contributions: B.I.K. and L.R.F. designed research; T.J.C., S.Q., and L.R.F. performed research; B.I.K. and L.R.F. analyzed data; and B.I.K. and L.R.F. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The trimer models have been deposited in the Protein Model Database, http://mi.caspur.it/PMDB [PMDB ID codes PM0075966 (2NWL-based model) and PM0075968 (2NWW-based model)].
This article contains supporting information online at www.pnas.org/cgi/content/full/0908570106/DCSupplemental.
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Citation Manager Formats
Article Classifications
- Biological Sciences
- Biophysics and Computational Biology


text:
Meet the family with an adventure playground in the garden
From garden adventure courses built on a shoestring to six-figure treetop hideaways, parents are pulling out all the stops to keep the kids off their screens this summer
"Our ninja garden is exciting and cool," says Sam, 9. "When my friends come round, they all head straight to the garden — which I like, because then they won't mess up my bedroom."
His brother, Joe, 7, is also a fan of the adventure playground their dad has created in their southeast London garden: "The ninja garden makes me very proud. We have a password for our treehouse that we change all the time. The treehouse balcony is our favourite place to have dinner."
Fans of the CBBC children's series The Dengineers will now what flights of fantasy can be turned into reality when kids get their way. Dens designed like an old-fashioned railway station, a 1960s American diner and an Australian outback shack


text: Demialia Levasseur 22 Posted July 18, 2017 Hello All I've been a resident of Secondlife almost 10 years. About 2 or 3 months after joining Secondlife I met the man who is now my RL and SL partner. We live together in RL with one of our SL daughters as a roommate. We have a homestead that I have set up like a farm/ranch, and we would really love to find a Horse to be our family horse. I've always wanted to own horses in RL but unfortunately so far that has not happened for me. You would have a loving and steady home... We are very down to earth and understanding people... Please feel free to contact me inworld or reply here if this sounds like something you are interested in. Thank you very much. Vivianna Rotaru (Demialia Levasseur) Quote Share this post Link to post Share on other sites


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The International Foundation for Autonomous Agents and Multiagent Systems (IFAAMAS) has selected a paper co-authored by Dr. Lewis Johnson of Alelo Inc., Prof. James Lester of North Carolina State University and the late Dr. Jeff Rickel of the University of Southern California to receive the 2017 Influential Paper Award. Entitled "Animated pedagogical agents: Face-to-face interaction in interactive learning environments," the paper published in the year 2000 laid the groundwork for a wide range of educational products that incorporate animated agent technology.Read more
Alelo CEO Lewis Johnson and NCSU Professor James Lester Receive Prestigious Award for Influential Paper on Pedagogical Agents
Alelo Launches the Enskill™ Digital Learning Environment
Alelo announced today its Enskill Digital Learning Environment, a system designed to deliver highly effective, economic learning solutions to close the skills gap faced by today's workers who need both technical and interpersonal skills to thrive while automation increasingly eliminates routine jobs.Read more
Alelo and Laureate International Universities to Co-develop Innovative Solutions for Learning Spoken English
Alelo Inc. (Los Angeles) and Laureate International Universities (Baltimore) have signed an agreement to co-develop computer-based modules for learning and assessing spoken English skills based on Alelo's virtual role-play technology and methods. The modules will be initially integrated into Laureate's online English-learning courses offered to its 50,000 lower-level students in Latin America.Read more
Alelo And Aptima Announced $3M Government Investment In Adaptive Language Learning Technology
The Office of the Secretary of Defense funded Alelo and Aptima to develop ALLEARN, an adaptive learning architecture that helps people learn foreign languages more rapidly and develop higher levels of proficiency. ALLEARN exploits advances in artificial intelligence to create rich interactive learning experiences that are optimized for each learner. Below is our interview with Dr. W. Lewis Johnson, President and CEO of Alelo.Read more
Alelo and Aptima Team to Develop Adaptive Language Learning Technology
Recognizing the unmet need for effective, relatively inexpensive foreign-language learning, the Office of Naval Research (ONR) has awarded a $3 million contract to Aptima Inc. and Alelo Inc. to develop ALLEARN, a system to accelerate foreign-language learning using artificial intelligence technologies.Read more


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An IoT smart mailbox for securely receiving parcels and getting notification on app without being present at home.
Monitor weather and security remotely with Blynk and the CC32000 LaunchPad!
Chunchunmaru is an Arduino UNO-based robot which can carry heavy loads (100kg-200kg) from one location to another.
Smart Garden is a plant environmental monitoring system.
This tutorial will help you to learn DHT11/DHT22 interfacing with nodeMCU and sending its data to Blynk Application.
A solution to measure noise level and sound pollution in urban areas.
saving water smartly.
In this project, we're going to make a keyless door lock system that uses a 4X4 keypad to enter the keys and a servo motor to open or close.
A cube with all the necessary sensors, suitable for a wide range of applications like agriculture. Know the land beneath you!
A new way to connect sensors! Connect up to five sensors to the particle powered WiSor and obtain real-time data on your mobile phone!
Particle Photon circuit allowing the display of environmental conditions (light, temperature, humidity, pressure) using the Blynk app.
A simple 4 channel design/program for monitoring orchid and plant moisture levels.
Use the Arduino101 Bluetooth wireless interface to read the Intertial Measurement Unit (IMU) with your phone or tablet using Blynk !
Controlling water and heat of a plantation, using real data as temperature, relative air humidity, and soil moisture.
Water is essential for life. Save water is saving Earth. So, why not use Blynk Board to save Earth?
Save time by knowing whether the Faculty is in his chamber or not.
Turn on a fan automatically with a Particle Photon using temperature sensing and the Blynk app.
Know when your clothes are dry
The IoT egg tray lets you know how many eggs you have left in the fridge and also shows temperature & humidity of your fridge.
The bird house will be outfitted with IOT to track what kind of conditions attracts guests and will tweet you when they are at the door!


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Global food production: From where?
Slide 8 by Jimmy Smith, director general of ILRI, of a presentation on 'Global health and sustainable food security: Why the livestock sectors of developing countries matter', at the Global Animal Health Conference on 'Developing global animal health products to support food security and sustainability' , 17−18 Oct 2013, in Arlington, Virginia (slide by ILRI).
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County council turns down chamber appeal
Brant county councillors have turned down an appeal to resurrect a city-county shared services committee.
"We already share more than 20 services," Ward 5 Coun. Joan Gatward said this week at a county council meeting. "The only thing that's really left is animal control and I don't think we need a joint committee to look at that."
Gatward made the comment in response to a letter from David Prang, president of the Chamber of Commerce of Brantford-Brant.
In the letter, dated April 9, Prang expresses disappointment over an earlier council decision. In March, county councillors unanimously rejected a city offer to resume shared-services committee meetings.
"Our chamber of commerce has long been a proponent of seeing cost savings and efficiencies at all three levels of government, which ultimately results in better value for all taxpayers," Prang said in his letter.
"The success of other joint-service agreements, such as the John Noble Home, ambulance services and social Services, demonstrates that when the City of Brantford and County of Brant work together to deliver services and programs, both communities benefit.
"We have a thorough understanding of the fiscal challenges being faced by all municipalities and encourage county council to pursue alternate service delivery methods with the City of Brantford … in an effort to hold the line on expenses while delivering the services that make our communities great."
Prang pointed out, in the letter, that the city is Brant County's closest and most populous neighbour.
County councillors voted to send a letter to Prang outlining their reasoning for rejecting the city's offer and highlighting the more than 20 services already shared by the two municipalities. The services include: the Business Resource Centre; the Brant County Health Unit; social services, which includes child care; economic development; andsome tourism initiatives.
The shared-services committee was originally established in March 2017 but its work came to an end prior to the October 2018 municipal election.
It was established to look at issues such as a regional transportation strategic plan, emergency services dispatch, animal control and library services.
Brantford city council voted unanimously in January in favour of re-establishing the committee. County councillors subsequently rejected the proposal, noting that, unlike the city, Brant has several other neighbours, including Oxford and Norfolk counties.
Instead of a shared services committee with the city, county officials are looking at ways of working with their other neighbours to deliver programs.


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"Colm can wait. Best to get some of them outta there. And much less fun to rob him and his score if he never finds out about it."
–Dutch van der Linde
Old Friends is a mission in Red Dead Redemption 2.
Mission overview
Looking for information on the O'Driscolls' planned train job, Dutch leads the gang into an attack on their camp.
Micah Bell, Bill Williamson and Lenny Summers have a small fight, before being stopped by Dutch van der Linde. Dutch, Arthur Morgan, Lenny, Javier Escuella, Bill, and Micah ride to the O'Driscoll Boys' camp to steal their plans to rob a train.
During the ride back to camp after slaying the O'Driscolls, they come across another O'Driscoll who they saw Colm reprimand. He takes flight, but Arthur chases him, hogties him, and brings him back to Colter to be imprisoned.
Gold Medal Objectives
- Catch and hogtie Kieran within 45 seconds.
- Kill 3 O'Driscolls in the same Dead Eye use.
- Complete within 15 minutes and 30 seconds.
- Get 15 headshots.
- Complete the mission without taking any health items.
- Numerous O'Driscolls - Killed by the Van der Linde gang after they discover their plan to rob a train.
Video walkthrough
- After capturing Kieran, if the player checks the map they will see six ally markers at the hideout, one for each of the participants in the mission as well as Hosea Matthews, although he has no involvement in this mission past brief appearances in the beginning and ending cutscenes.
- If you spare the O'Driscoll in the first mission Outlaws from the West, he will appear in the cabin in front of you after you and Dutch get in cover.
Community content is available under CC-BY-SA unless otherwise noted.


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Our Wish List
Donate to our Transformational Housing Program
Our Transformational Housing Program is for motivated pregnant women and single mothers with children ages 12 or younger. When they come to us, they often have nothing but the clothes on their backs. We need your help to provide them with the basic personal care products and household items they need.
We observe the following drop off hours at our Norwell Home (12 Concord Street in Nashua):
- Saturdays from 9 a.m. – 12 p.m.
- Sundays from 11 a.m. – 1 p.m.
- Weekdays by appointment only to ensure staff availability.
Questions about donating items? Contact case manager Kelsey Sanborn at firstname.lastname@example.com or 603.816.0281.
Amazon Wish Lists
The easiest way to identify needs and donate is to visit our wish lists on Amazon. Simply add any items from the lists to your cart and have them delivered directly to us.
Other Items Needed
Household Items
- Furniture in good condition (no mattresses) — Please note we are unable to accept furniture without prior approval.
- Cleaning supplies — Laundry detergent, paper towels, Lysol or all-purpose cleaner, bleach, dish soap, trash bags, etc
- Mops, brooms and vacuums
- Twin bedding for women and children; crib sheets and toddler bedding sets
- Pillows — New only
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- Small kitchen appliances – New and gently used
- Kitchenware — New and gently used pots and pans, silverware, glasses, dinnerware
- Kitchen Storage — Plastic containers, wraps and bags
Personal Care
- Diapers and wipes
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- Common over-the-counter medications for adults and children
- Toiletries — Hair care products, body wash, lotions, etc.
- Thermometers and basic first aid items
- Toilet paper and tissues
Birthday Party Supplies for Children
Wrapping paper for various ages
Party decorations and supplies such as napkins, plates, etc.
Spring/Summer Items
- Kids' Bathing Suits
- Bug Spray
- Sunscreen
- Umbrellas & Raincoats
Fall/Winter Items
- Hats, mittens and gloves
- Winter coats
- Snow toys, i.e. coasters, sleds, skates


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Islamabad, April 4 (IANS) Dost Muhammad Khosa, Pakistani Punjab's commerce and investment minister, resigned Wednesday following a case being registered against him after an actress whom he had married went missing, a media report said.
Khosa, who is also a former chief minister of Punjab, resigned over the Sapna missing case, Geo News cited sources as saying.
In December last year, Khosa was booked for the kidnapping of actress Sapna whom he had married.
The police registered the case against Khosa and his accomplices following a court order.
A petition had been filed in Lahore High Court for the recovery of the actress.
The petitioner had said that Sapna has been missing for eight months and police were not supporting them.
The family feared that Khosa, who had married Sapna, has been killed.
Khosa had admitted in the High Court that he had married Sapna but later divorced her. He said he did not know about her whereabouts.
Read More: Delhi High Court | Khosa Dal Singh | High Court | Allahabad High Court | Sultanpur Dost | Gujrat High Court | High Court Bulding Po | Barnes High School | High Court Building | New High Court | High Court Bilaspur | High Court So | Royapettah High Road | Perambur High Road | Nungambakkam High Road | Palayankottai High Grounds | High Forest | Ernakulam High Court | Talcher High School | Patna High Court
Programming tutorials
RoseIndia.Net is offering online tutorials in many programming technologies. Learn Java, JSP, EJB, JDBC, Servlets, Hibernate, Spring, Big Data, BioInformatics, Java Server Faces, Struts, XML, MySQL, Web development, PHP and other Technologies. Online training offers: Best Online Training for Big Data and Hadoop, Big Data and Hadoop Training, Big Data and Hadoop Training in Delhi and Big Data and Hadoop Training - Online and classroom. Take advantages of these training course and become master in Big Data industry. Check the latest Google News Architecture. Check the services provided by IoT(Internet of Things) Application Development Companyin India.


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This Orzo Corn Salad with Cucumber Feta Dressing makes a great summer side.
I put a bit of a Mexican twist to it with jalapeno peppers and red pepper.
Chef's Tips for Orzo Corn Salad
- If it is corn season you may want to steam fresh corn and then cut the kernels off the cob. 1 ear of corn generally produces about 1 cup of kernels.
- If using frozen or canned corn -don't cook it. Just defrost or drain according to which one you use and add to the salad.
- You can make this as spicy as you like by retaining the jalapeno seeds and membrane for hotter and removing them for milder.
|Prep Time
|10 minutes
|Cook Time
|9 minutes
- 1/2 cup crumbled feta divided 1/4 +1/4 cup
- 2 cups cucumber divided 1 cup + 1 cup
- 2 Tbsp olive oil
- 1 lime-zest and juice reserve the zest for garnish on top of the salad
- 3/4 cup uncooked orzo
- 2 cups sweet corn kernels steam fresh corn and cut kernels off the cob or use frozen or canned corn without cooking
- 1 medium jalapeno pepper
- 1/4 cup sweet red pepper
- Peel and seed the cucumber. Cut into 1" cubes. Add 1 cup to the blender for the dressing and reserve the other cup for the salad.
- Add 1/4 cup of feta (reserve remainder for the salad) to the blender. Add 1 cup of cucumber, oil and lime juice in blender and blend until relatively smooth.
- Cook the orzo according to the package instructions. (Mine cooks for about 7 minutes). Drain and rinse.
- Mince the jalapeno pepper. (Remove seeds and white membrane if you don't want it to be too hot).
- Mince the red pepper.
- Add the orzo, corn, cucumber, feta, jalapeno and red pepper to ad large bowl. Stir to mix. Pour dressing over and stir again. Sprinkle the lime zest on top of the salad.
- Serve immediately.
Recipe Notes
Nutrition Facts
Orzo Corn Salad with Cucumber Feta Dressing
Amount Per Serving
Calories 201 Calories from Fat 63
% Daily Value*
Total Fat 7g 11%
Saturated Fat 2g 10%
Polyunsaturated Fat 0.1g
Monounsaturated Fat 0.04g
Cholesterol 7mg 2%
Sodium 179mg 7%
Potassium 133mg 4%
Total Carbohydrates 29g 10%
Dietary Fiber 2g 8%
Sugars 3g
Protein 6g 12%
Vitamin A 6%
Vitamin C 24%
Calcium 3%
Iron 7%
* Percent Daily Values are based on a 2000 calorie diet.


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Hundreds of flights disrupted amid thick fog
Hundreds of flights serving London airports were cancelled or delayed amid thick fog.
By noon some 62 flights to or from London Heathrow on Tuesday were cancelled and a further 380 were delayed by more than 15 minutes.
That means more than one in three flights were disrupted.
British Airways alone saw 45 cancellations and 237 delays at the west London hub, which is the UK's busiest airport.
London City saw 62 cancellations and 46 delays, while Gatwick experienced no cancellations but 173 delays.
The figures were published by aviation data firm FlightStats.
Fog reduces visibility for pilots and air traffic controllers, meaning the safe distance between aircraft is increased. This disrupts the normal pattern of take-offs and landings.
A Heathrow spokeswoman said: "This morning's heavy fog has led to reduced visibility across all London airports which may result in some passengers experiencing disruption to their journeys today.
"Passengers are advised to check their flight status with their airline before coming to Heathrow. As always, our top priority is the safety of passengers and we apologise to those whose travel has been affected by today's weather."
The conditions also caused delays to trains serving Gatwick.
A statement on the National Rail Enquiries website read: "Southern and Thameslink have received a number of reports from drivers that due to heavy fog they are having to run at a reduced speed due to reduced visibility.
"When thick fog is present, drivers are unable to see signals from their normal distances and to continue to operate safely they must drive at reduced speeds so that they are able to view signals."
The derailment of a Great Western Railway train in West Ealing at around 7am affected trains serving Heathrow.
One of the lines was blocked, causing delays and cancellations to London Paddington-bound services from Heathrow and Slough.
The line was reopened around 60 minutes later but disruption was expected to continue until 6pm as services recover.
A Network Rail spokesman said: "Our engineers are on site and will clear the derailed train as soon as possible."
Meanwhile, the UK is braced for a blast of gales and heavy rain as Storm Diana sweeps in from the Atlantic.
The Met Office has issued severe weather warnings from 3am on Wednesday until 3pm on Thursday as gusts of up to 80mph could hit western parts of the country.


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"Six years ago, Wisconsin's Gundersen Healthcare System set a goal to reduce energy consumption and rely more on renewable energy. On October 14, Gundersen, which makes up a network of hospitals, medical clinics, nursing homes and other health facilities, did more than reach that goal — it successfully produced more energy than it consumed, a milestone it's kept up every day since the 14th.
The health system, which is the first of its kind to become fully energy independent, announced the success of its environmental and efficiency efforts on Monday. In its quest to achieve energy independence, Gundersen has saved nearly $2 million each year from energy conservation and efficiency efforts.
Reaching energy independence isn't easy for any building, but it's especially challenging for hospitals, which can use up to two and a half times as much energy as commercial buildings, due to high tech machinery that often needs to be kept on 24-hours a day.
"We did not set out to be the greenest health system," Gundersen CEO Jeff Thompson said in a statement. "We set out to make the air better for our patients to breathe, control our rising energy costs and help our local economy. We believe we have made more progress on all three than anyone else in the country."
Gundersen relied on savings and government grants to complete its sustainability projects, and according to Jeff Rich, executive director of Gundersen's environmental program, the health system only invested in projects that were "two-sided green — green for the environment and green for finances."
Energy initiatives:
Solar PV, Biomass bioler, Waste reduction, Cogeneration, Methane Gas from Landfill
Full article: http://thinkprogress.org/climate/2014/11/11/3591150/green-hospital-system/
Like us on Facebook: https://www.facebook.com/freemanapplegate?ref=aymt_homepage_panel


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Additional information
|2.3 oz
Inspiring Creativity and Encouraging Handmade
1 6×8 Stamp Set with 31 Images
Create beautiful floral bouquets with these hand-painted flowers. The look of watercolor without the work.
Use a variety of inks to create unique looks and you can emboss the top layer for some added interest and a little sparkle.
Have fun creating your own arrangements. You'll love this set. Coordinating Dies available.
|2.3 oz