text:
Edit your search:(X) Davis, CA
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ACCOUNT MANAGER (new position)
Gaynor Telesystems, Inc. - Davis, CAOUTSIDE ACCOUNT MANAGER Northern California We are currently looking to add to our team a seasoned account manager with strong lead generation skills, time management and exceptional customer service. We have a strong team with an impeccable reputation. For this unique opportunity we will on ...
Posted: 2017-02-24 | Industry: Tele Equipment | Base Salary: At least $20,000
Territory Manager - Sales Rep. / Trade Marketing
RJ Reynolds - Davis, CATerritory Managers / Trade Marketing Representatives are assigned to a geographic territory where they are responsible for: Calling on established traditional and nontraditional retail outlets Selling and executing national/local brand building marketing programs and initiatives within these acco ...
Posted: 2017-02-23 | Industry: Food and Beverage | Base Salary: At least $40,000
Kitchen Table Closer - No cold calling
The Monteith Group - Davis, CARated "Top 10 company to work for" byExperience Incmagazine rated us one of the "Fastest growing companies in America" Entrepreneur & CultureIQawarded us "Top Culture" Due to site guidelines - we can email you our5 Min. videoof, Who we are and What we do, once you've applied. We are looking for s ...
Posted: 2017-02-15 | Industry: Insurance | Base Salary: 100% commission
Territory Manager - Sales Rep. / Trade Marketing
RJ Reynolds - Davis, CATerritory Managers / Trade Marketing Representatives are assigned to a geographic territory where they are responsible for: Calling on established traditional and nontraditional retail outlets Selling and executing national/local brand building marketing programs and initiatives within these acco ...
Posted: 2017-02-11 | Industry: Food and Beverage | Base Salary: At least $40,000
Sales Professional- Tired of Developing Business - Just c ...
Sears Home Improvement - Davis, CAImmediate Hire For immediate consideration please apply...Immediate Interviews Held Sears Home Improvement Division- Now Hiring Outside Professional Sales-Class to start 3rd week of June Please complete the application: [please apply online] for an Immediate Interview. This is a real opportun ...
Posted: 2016-07-14 | Industry: Real Estate | Base Salary: 100% commission
Outside Sales Representative
MOBILE AUTO GLASS - Sacramento, CAAuto glass Company looking to hire full time outside sales representative to market to reputable insurance offices in the Sacramento area. Successful candidate will be assigned to establish accounts to represent the company, and develop business relationships. Applicants must have excellent commu ...
Posted: 2017-02-22 | Industry: Automotive | Base Salary: Yes (amount not posted)
Residential Sales Consultant
Protection 1 - Sacramento, CAJob Overview: If your experience is in presenting products and services to consumers Protection 1s Residential Security Consultant role could be the right choice for you! As security consultant you will be responsible for initiating contacts, networking and following up on leads to present and ...
Posted: 2012-12-21 | Industry: Security | Base Salary: 100% commission
EXPERIENCED APPOINTMENT SETTERS APPLY NOW! $45K - $55K 1s ...
Company confidential - Sacramento, CAWe are seeking an EXPERIENCED APPOINTMENT SETTER to connect people in pain with local medical professionals. This is a unique, full-time position with FLEXIBLE SHIFTS and HIGH EARNING POTENTIAL. If you are interested in a STABLE and REWARDING SALES CAREER with a growing company, then this is the ...
Posted: 2017-02-24 | Industry: Work from Home | Base Salary: Yes (amount not posted)
Business Development Represenative - Lead Generation
Wheelhouse Enterprises - Sacramento, CABusiness Development Representative Who is Wheelhouse looking for? We are seeking high energy, top performers that are driven by the desire to make money! The Business Development Representative (BDR) is responsible for transitioning customer interests into qualified leads for world class B2B t ...
Posted: 2017-02-24 | Industry: Work from Home | Base Salary: At least $20,000
Seeking Telesales Professionals - Career-Level Income - A ...
Company confidential - Sacramento, CAWe are looking for an ASSERTIVE PHONE SALES PROFESSIONAL to sell a FREE exam and consultation with a local licensed medical provider to individuals in need of treatment. This is a full-time Inside Sales position with high earning potential. ABOUT THE POSITION: All leads are provided via predictiv ...
Posted: 2017-02-23 | Industry: Work from Home | Base Salary: At least $40,000
Jobs 1-10 of 62 (Next 10)


text:
Twist the bacon for a texture that's crisp on the ends and chewy in the center.
Makes 6 servings
+ Add To Shopping List
1 pound hickory-smoked bacon slices
1 cup firmly packed light brown sugar
1 tablespoon cracked black pepper
Prep: 15 Minutes
Bake: 25 Minutes
Cut bacon slices in half.
Combine sugar and pepper in a shallow dish. Dredge bacon in sugar mixture, shaking off excess.
Twist each bacon slice, if desired. Place bacon in a single layer on a lightly greased baking rack in an aluminum foil-lined baking pan.
Bake at 425° for 20 to 25 minutes or until crisp. Allow bacon to cool before serving.
Created date
October 2005

subset: commoncrawl


text:
You've got to login or join dailymile to do that
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Tracking my way back to 235.
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text: We had been hiking along the beach trail and had lunch here. We were two couples and the ladies turned back first so had a head start on us. A great beach location and lots of choices on their menu. All of us enjoyed our choices. A great place to have a drink by the water.


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"One more small alley" Top 5 Page for this destination Pula Things to Do Tip by JLBG
Pula Things to Do: 252 reviews and 508 photos
From Ulica Sergijevaca towards Ulica Castropola and the castle are very steep, which in the full heat of summer is not very appealing. It would be wiser climb the long way, as explained in another tip and walk down one of this charming alleys !
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Nitrates in Ground Water
IAH Selected Papers on Hydrogeology Volume 5
Edited by Lidia Razowska-Jaworek Andrzej Sadurski
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Nitrates in Ground Water by Lidia Razowska-Jaworek
Book DescriptionThe first book devoted entirely to the problem of nitrate risk and behaviour in groundwater, this volume includes twenty-seven papers selected from those presented during the Euromeeting of the International Association of Hydrogeologists "Nitrate in Groundwater in Europe" held in Wisla, Poland in 2002. The problems presented and discussed in Wisla covered all aspects of nitrate pollution of groundwaters globally, while the articles describing the case studies focus on groundwater in Europe. The remaining chapters examine the main chemical and physical processes which affect nitrate in groundwater, as well as describing nitrate distribution in various aquifers, seen as an essential problem areas of groundwater protection.
Buy Nitrates in Ground Water book by Lidia Razowska-Jaworek from Australia's Online Bookstore, Boomerang Books.
Book DetailsISBN: 9789058096647
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Publish Date: 15-Aug-2004
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With student loan debt in the U.S. now well beyond $1 trillion, everyone seems eager to get into the debt-reduction business. Some cities will pay down your debt if you move there, while a growing number of employers are making loan payment contributions part of the benefits package. Now some financial institutions are dangling the debt-reduction carrot in front of potential customers — but should you bite? [More]
Banks Trying To Attract Customers With Products To Reduce, Repay Student Loans
20,000 For-Profit College Students Ask Education Dept. To Cancel Their Student Loans
As the fallout continues from the collapse of Corinthian Colleges Inc. — former operator of Everest University, WyoTech, and Heald College — the Department of Education is trying to sort through nearly 20,000 loan-forgiveness requests from former students who claim that CCI and other for-profit colleges misled them into taking out huge student loans. [More]
Screwed Over By A For-Profit College? You Probably Signed Away Your Right To Sue
When Corinthian Colleges Inc. collapsed, leaving thousands of students in the lurch with student loan debt and credits that they didn't know would be usable at other schools, they were generally unable to sue the failed for-profit educator because the students had unwittingly signed away their right to a jury trial or class action. CCI wasn't the only for-profit operator with this anti-consumer practice, and a new report tries to get a grasp on the scope of the problem. [More]
Feds Will Notify Disabled Borrowers Eligible For Student Loan Discharge
Under the law, borrowers who are permanently disabled are eligible to have their federal student loans discharged. While the government has taken steps in the past to make the process more streamlined for consumers, the Department of Education will now proactively seek out eligible borrowers. [More]
Feds Shut Down Student Loan Debt Relief Operation That Collected $3.6M In Illegal Fees
Federal law bars debt relief services from receiving upfront fees before they've even renegotiated a single debt for a customer. But one student loan debt relief operation allegedly took in nearly $3.6 million in illegal fees, only to enroll borrowers in programs that are already available for free.
Sallie Mae To Offer Increasingly Popular Parent Student Loans
For years, parents have assisted their children in shouldering the increasingly high cost of college: co-signing private student loans, taking part in federal loan programs, and saving for years to contribute. Private student loan lenders have been offering parent loans as an alternative for several years. And now the country's largest lender plans to enter the fray with its own version. [More]
Former Corinthian College Students To Have Easier Time Getting Student Loan Discharges
For nearly a year, advocates and lawmakers have shared their dissatisfaction with the Department of Education's pace and complicated process on discharging the student loan debt for students of now-defunct for-profit chain Corinthian Colleges. Today, the Department is expected to clear a path for debt relief for these students after determining that state investigations into the schools found enough proof that the company widely misled students about their futures if they attended the schools. [More]
Are Employee Student Loan Contributions The Next 401(K)?
Any help graduates can get when it comes to repaying their mountains of student loan debt is often welcome: from cities offering debt forgiveness to keep young adults in their areas to programs in which states will pay for portions of their education. Now, more employers are jumping on the assistance train by providing student loan payment contributions as part of their compensation plans. [More]
Show me someone who supports robocalls, and I'll show you someone that has very few friends. Which is why it's baffling that the Senate has yet to act on a bill introduced last fall that would close a loophole allowing the government to make debt-collection robocalls. But you know who does support robocalls? The student loan companies that are currently trying to convince Congress that these invasive annoyances are really for our benefit. [More]
Student Loan Companies Could Face Enforcement Actions Over Automatic Defaults
In recent years, countless private student loan borrowers have found themselves placed in automatic default – even if they were up-to-date on payments – when their co-signer died or filed for bankruptcy. Federal regulators now appear poised to rein in this often devastating practice, warning student loan lenders and servicers that they could soon face enforcement action if they continue the practice. [More]
Man Claims He Was Arrested For Unpaid Federal Student Loan Debt
More than a century ago, the U.S. did away with the idea of debtor's prison, and the Fair Debt Collection Practices Act explicitly forbids debt collectors from using the threat of jail time as a way to collect on owed money. So how does a Texas man end up with U.S. Marshals at his door, ready to arrest him for a nearly 30-year-old student loan debt? [More]
Dept. Of Education Working On Rules For Defense Of Repayment Law After Influx Of Claims
Under federal law, student loan borrowers may be eligible to have their debts discharged if they prove the school they attended deceived them with false promises related to their future careers. However, the measure has been used only sparingly in the past and few clear rules outline the forgiveness process. Now, after nearly two decades on the books, federal officials are finally getting around to crafting rules that could remove one roadblock for students seeking relief. [More]
New York Program Will Pay For Two Years Of Recent Graduates' Student Loans
For many recent graduates, repaying their education debt obligations can be a struggle. For some in New York, that struggle just got a little less cumbersome thanks to a recently launched student loan forgiveness program that aims to help the debtors land on their feet after graduation.
Now that 2015 is done and we finally learned that Luke Skywalker is actually Faye Dunaway's daughter (and sister!), it's time to take off the party hats, sweep up the confetti, and do the walk of shame forward into the uncharted territory of the year to come. [More]
While 2015 didn't smash as many records for "terrible things that can happen to consumers in a single event" as 2014 — what with last year's GM recall, Sony hack, and the like — we still had rather a lot go on in the last 365 days. Here's a run-down of the numbers from 2015.
7 Things We Learned About Federal Student Loans & The Companies That Profit From Them
Fifty years ago, Congress created the federal loan program as a way to help Americans realize their dreams of a better life through higher education. While millions of students have no doubt benefited from the program, millions of others have found themselves burdened by mountains of debts, fielding calls from debt collectors and loan servicers, and watching as their paychecks are whittled down by garnishments. Today, seven million former college students are in default with a record $115 billion in federal loans. While those figures may be oppressing borrowers, it's providing a stream of income – and profit – for companies contracted by the government to collect payments from debtors. [More]


text:
craig's list scrapper for leads
This project received 5 bids from talented freelancers with an average bid price of $75 USD.Get free quotes for a project like this
Project BudgetN/A
Total Bids5
Project Description
basic coding. I need someone to build a craig's list scrapper that'll help me scrape names, emails, and numbers of specific home sellers like FSBO or landlords renting houses.
Skills Required
Looking to make some money?
- Set your budget and the timeframe
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text:
This document outlines the Interledger architecture and explains how the different layers relate to each other.
The Interledger architecture is heavily inspired by the Internet architecture described in RFC 1122, RFC 1123 and RFC 1009.
This is a strawman proposal. Choices made in this document are merely intended as a starting point.
This document is intended to provide an overview over the Interledger architecture and protocol suite.
All electronic transfers have to be recorded in stateful systems. Otherwise the same instance of an asset could be sent to two different destinations, essentially duplicating the asset. This is also known as a double-spend. We call these stateful systems ledgers.
Ledgers consist of accounts. Accounts are individual buckets containing a decimal amount of one type of asset, usually (but not always) associated with an owner. This amount is also called the account's balance. Balances can be positive or negative, representing assets or liabilities.
Assets can be transferred between accounts on the same ledger. We call these events book transfers or just transfers.
A transfer of assets across ledgers requires two or more local book transfers. Some system must know the relationship between the two transfers. We call this system a connector. The same system may act as both a ledger and a connector.
The Interledger is a network of independent and diverse ledgers. Each account is part of a particular ledger; the Interledger itself is only conceptual.
We assume that all assets within one ledger are fungible. Within one ledger transfers can happen directly from sender to recipient and do not require a third party exchanger.
When a single organization such as a bank manages accounts in multiple different assets of different types, we treat each asset as belonging to its own ledger.
Just as Internet gateways do not keep connection state information, connectors do not keep transfer state information. All state is kept at the ledger level and connectors merely react to and trigger ledger events.
Note that this does not mean that all connectors are necessarily stateless. They may keep track of their available liquidity or even maintain an internal ledger if they are transacting on behalf of third parties. But we are assuming that they do not keep authoritative state about transfers.
Routing is a complex and difficult problem, and ought to be performed by the connectors, not the end users of the system. An important objective is to isolate user software from changes caused by the inevitable evolution of the Interledger routing architecture.
A basic objective of the Interledger design is to tolerate a wide range of ledger characteristics --- e.g. throughput, latency, fees, access restrictions, reliability, decentralization. In addition it must support all types of different asset characteristics --- e.g. divisibility, fungibility, interest/demurrage, issuance.
The Interledger architecture is separated into four layers:
The application layer is the top layer of the Interledger protocol suite. Protocols on this layer are responsible for negotiating the key properties of a payment:
Once these parameters are decided, the application layer protocol will instantiate a transport layer protocol to initiate the payment.
Application layer protocols include open protocols such as the Open Web Payment Scheme (OWPS) and proprietary protocols such as the Ripple Payment Services Protocol (RPSP).
The transport layer provides end-to-end transaction services for applications. There are three primary transport layer protocols at the moment:
OTP is a trivial transport protocol which simply transfers funds without the protection of escrow. Hence it is only suitable for very small microtransactions where efficiency matters more than reliability.
UTP uses escrowed transfers to guarantee that no funds leave the sender's account unless the transaction was successfully completed. It is suitable for transactions of any size and should be considered the default choice.
ATP uses escrowed transfers and a set of pre-agreed, trusted notaries in order to guarantee atomicity across multiple ledgers. It is relatively expensive to set up, but can be used to reduce the need for reconciliation.
All Interledger transport protocols use the Interledger Protocol (ILP) to communicate with connectors about transfer requests. This may include requesting a quote or requesting a transfer on another ledger.
The Interledger layer defines a standard way to refer to ledgers, accounts and amounts. This is used in routing as well as to try and make quotes comparable.
In order to facilitate transfers between accounts, ledgers must implement some API or protocol. We call this the ledger layer. There is a wide variety of ledger layer protocols, corresponding to the many different types of ledger.
Ledger protocols are responsible for executing the individual transfers that constitute an Interledger transaction. They are also used by connectors to communicate with each other. Ledger layer protocols can differ widely depending on the type of ledger. For example, a central ledger will likely use a very different protocol than a blockchain, but for interledger purposes they are both ledgers and may be accessed using the same primitive operations as defined in this architecture.
For minimal Interledger support, the ledger MUST have the ability for funds to be transferred from one account to another. This enables OTP transactions to pass through this ledger. It also allows third parties to act as escrow providers in order to enable UTP and ATP transactions on the ledger.
For basic Interledger support, a ledger MUST fulfill the requirements for minimal support and also the following:
The ledger MUST provide cryptographic escrow. Escrow means that transfers can have four states:
Hint: Escrow is the financial equivalent of a two-phase commit.
The ledger MUST be able to verify a simple SHA-256 hashlock cryptographic escrow condition in order to automatically trigger the release of the escrow. It must also allow a timeout to automatically roll back an escrowed transfer.
The ledger MUST support attaching a short message or memo to each transfer.
The ledger MUST fulfill the requirements for basic support and also the following:
It MUST support all cryptographic condition types with the status "recommended".
It MUST support memos to be added for the credited and debited accounts separately and SHOULD support fairly large memo sizes.
It MUST support a way to discover connectors connected to the ledger.
It MUST support a way to look up a fulfillment by condition hash. It SHOULD automatically reject new transfers (that have not been prepared yet) that have an execution condition for which the ledger already knows the fulfillment. This aids in UTP error recovery.
Simple Ledger Protocol (SLP) is a RESTful, JSON-based protocol that was developed specifically to provide the minimum functionality required for full Interledger support.
A reference implementation of a ledger using SLP can be found here.
Blockchains are distributed, peer-to-peer systems that provide consensus over a single shared state. Any blockchain that supports escrowed funds transfers is in principle capable of acting as a ledger connected to the Interledger.
Bitcoin for instance supports multiple credits and debits as well as SHA-256 hashlocked escrow transfers which means it can participate in OTP/ILP and UTP/ILP Interledger transactions. Bitcoin's BIP-65 enhancement proposal provides the timeouts required for Basic level support.
Legacy protocols often do not provide escrow functionality. In this case, the protocol can either be upgraded, or a highly trusted participant (e.g. a bank) can act as an escrow provider.
There are large numbers of proprietary ledgers out there. This includes web-based and mobile wallets. These types of systems can usually be extended with cryptographic escrow functionality by their operator in order to connect them to the Interledger.
Some proprietary protocols intentionally do not provide general ledger functionality. A common example are stored-value systems, such as gift cards, loyalty points or pre-paid accounts. Such systems can still be connected to the Interledger in a limited capacity. For example a pre-paid account ledger could be set up to act as a receiving ledger, but not as a sending or intermediate ledger.
By creating two classes of users -- resellers and end users -- and only allowing transfers if the sender is a reseller and the recipient is a user, merchants can create Interledger-capable ledgers which behave like stored-value systems. Such systems allow deposits, but do not allow withdrawals.
The Interledger Protocol (ILP) ensures that different connectors are interoperable and can work together to route transactions.
When initiating an Interledger transaction, the sender will make a transfer to a connector using their local Ledger layer protocol. Within this transfer, the sender will include an ILP Packet which tells the receiving connector the final destination, the amount to be transferred and -- if applicable -- the condition.
Note that the exact method of transmitting this data packet is dependent on the ledger layer protocol. Typically, it will be included in the transfer in a memo field. However, some ledgers may specify a different method for transporting the ILP packet.
Before an Interledger transfer takes place, the sender will request quotes from connectors which are connected to the same ledger. These quote requests happen via the Interledger Quoting Protocol (ILQP).
Senders MAY cache quotes and send repeated transfers through the same connector.
The Interledger Control Protocol (ILCP) is a protocol used by connectors to exchange routing information and communicate payment errors.
TODO: add link
Transport layer protocols are responsible for coordinating the different transfers that make up an Interledger transaction. The safety guarantees afforded to the participants of a transaction vary depending on the type of transport protocol used.
The Optimistic Transport Protocol (OTP) is the trivial case of a transport protocol. It simply makes a transfer to the next connector. The connector may or may not make the requested transfer and the sender is out the money either way.
For most cases, OTP will not be appropriate. However, it may make sense in the case of recurring microtransactions where a few lost transactions are not a big deal.
TODO: Need to figure out how OTP can detect dropped transfers and adjust the route accordingly.
The Universal Transport Protocol (UTP) is the standard and recommended transport protocol. It sets up a cascading chain of escrowed transfers in order to ensure delivery of funds.
The protocol flow is described in the Interledger whitepaper.
A UTP execution chain can be interrupted if a connector fails to deliver the condition fulfillment. In that case the sender will think that the transaction failed and retry it. While retrying, some connector will try to create a prepared transfer on a ledger which already knows the corresponding fulfillment, which will cause the request to fail. The connector will notice this and simply pass back the fulfillment without paying anything.
In other words, the failed connector will lose money and some other connector will win money, but from the sender and recipient's point of view everything has executed normally.
The Atomic Transport Protocol (ATP) is the most conservative, but also most complex of the standard Interledger transport protocols. In addition to the ledgers and connectors, it also involves a set of impartial notaries, which must be agreed upon by the sender, recipient and connectors involved in the transaction.
Since each party chooses their own set of notaries to trust, there may not be any overlap and ATP could not be used in that case. Notaries may be selected using an automatic process or preselected by a group with standing agreements among the participants.
The protocol flow is described in the Interledger whitepaper.
ATP can be used as a sub-protocol for part of a UTP transaction. This happens when a connector decides to create an ATP transfer as the next hop. It takes on both the risk of being unable to pass on the receipt and the risk of the notaries failing. This makes sense to do if the next connector quotes a better price for an ATP transfer than for a UTP transfer.
Similarly, an ATP transaction can turn into a UTP transaction when a connector decides to use UTP for the next hop. It takes on the risk of not being able to pass on the receipt to the notaries. This makes sense if ATP is not available.
Application layer protocols deal with the exchange of payment details and associated negotiation.
The Simple Payment Setup Protocol (SPSP) is an application layer protocol for negotiating payment details. SPSP handles account and amount discovery, condition creation, quoting and setup. SPSP uses Webfinger (RFC 7033) and an HTTP-based protocol for querying account and amount details, ILQP for quoting, and UTP for payment execution.
The protocol is described in IL-RFC 9.
Creators of other application layer protocols should consider the following:


text:
Free personalized radio that
plays the music you love
Air (Sarabande) In E Major, Hwv 425: Handel Sarabande And Variations
Features of This Tracka supremely inspired melody
a well-known composer
tonal harmony
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and many other similarities identified in the Music Genome Project
These are just a few of the hundreds of attributes cataloged for this track by the Music Genome Project.show more
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This study investigated the metabolic requirements for neuronal progenitor maintenance in vitro and in vivo by examining the metabolic adaptations that support neuronal progenitors and neural stem cells (NSCs) in their undifferentiated state. We demonstrate that neuronal progenitors are strictly dependent on lactate metabolism, while glucose induces their neuronal differentiation. Lactate signaling is not by itself capable of maintaining the progenitor phenotype. The consequences of lactate metabolism include increased mitochondrial and oxidative metabolism, with a strict reliance on cataplerosis through the mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) pathway to support anabolic functions, such as the production of extracellular matrix. In vivo, lactate maintains/induces populations of postnatal neuronal progenitors/NSCs in a PEPCK-M-dependent manner. Taken together, our data demonstrate that, lactate alone or together with other physical/biochemical cues maintain NSCs/progenitors with a metabolic signature that is classically found in tissues with high anabolic capacity.
Glucose is still considered the major fuel for the adult brain, even though lactate signaling or utilization by neurons has been commonly observed both in vitro and in vivo (Bouzier-Sore et al. 2003; Gallagher et al. 2009; Wohnsland et al. 2010). This is especially the case in the developing brain, where lactate is a major substrate for oxidative metabolism in addition to being selectively utilized as an anabolic source for cell proliferation and differentiation (Bolanos and Medina 1993; Nehlig and Pereira de Vasconcelos 1993; Medina et al. 1996; Medina and Tabernero 2005; Zilberter et al. 2010). In neurons, lactate is transported across the cell membrane by the monocarboxylate transporter 2 (MCT2) (Pellerin et al. 1998; Debernardi et al. 2003) and subsequently converted to pyruvate, which can be further oxidized or used as an initial substrate in anabolic pathways. In contrast, astroglial cells release lactate to the extracellular milieu through the transporters MCT1 and MCT4 (Rafiki et al. 2003; Pierre and Pellerin 2005). Lactate is also involved in the maintenance of brain energy turnover and neurovascular coupling, by regulating cyclic AMP formation through GPR81 receptor activation, a process independent of lactate metabolism (Bergersen and Gjedde 2012; Bozzo et al. 2013; Lauritzen et al. 2013).
Tissue remodeling and regeneration depend on adult stem cells and their progenitor properties, which are mostly regulated by the cellular microenvironment or niche (Scadden 2006; Ivanovic 2009; Nakada et al. 2011; Gattazzo et al. 2014). Neural stem cells (NSCs) of the central nervous system reside in niches that are in close contact with the vasculature (Shen et al. 2008; Tavazoie et al. 2008; Goldman and Chen 2011). These neurogenic neurovascular niches contain a extracellular matrix (ECM) rich in laminins, type IV collagen, nidogen, and highly glycosylated sulfate proteoglycans (Yanagisawa and Yu 2007; Miner 2008; Shen et al. 2008; Nasu et al. 2012). The balance between cellular self-renewal and differentiation is mediated by cell adhesion to the ECM, and by local cytokines, systemic hormones, and oxygen and nutrient supplies (De Filippis and Delia 2011; Lehtinen et al. 2011; Nakada et al. 2011; Ziegler et al. 2012).
Our previous work showed that neuronal cultures grown on lactate-releasing biomimetic materials composed of poly-l/dl-lactate or treated with l-lactate were maintained in a less differentiated state in which a significant pool of neuronal progenitors was preserved (Alvarez et al. 2013). Our group has also shown that the biophysical properties and topography of the supplied substrate are important for NSC and glial progenitor maintenance (Mattotti et al. 2012; Alvarez et al. 2014). In this study, we explore the role of lactate in the NSC niche by examining the differential effects of lactate and glucose on cellular fate, metabolism, and anabolic capacities of neural progenitors in vitro and in vivo. Our results provide evidence that lactate intake through MCT2 and its subsequent oxidative metabolism direct progenitor commitment to a neuronal progenitor fate. The consequences of lactate metabolism are increased mitochondrial oxidation and a dependence on cataplerosis through the mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) pathway. PEPCK-M enhances tricarboxylic acid (TCA) cycle flux and phosphoenolpyruvate (PEP) export to feed-forward carbons into the cytosolic 3-carbon pool, thus effectively promoting the cellular anabolic potential and ECM production. Taken together, these results indicate the coexistence in the neurovascular niche of NSC and progenitors with different metabolic signatures.
Materials and Methods
Intraventricular Brain Injections in Postnatal Animals
All animal housing and procedures were approved by the Institutional Animal Care and Use Committee of our institution in accordance with Spanish and EU regulations.
The brains of newborn (postnatal day 0, P0) mice were injected in the lateral ventricle with 2 µL of lactic acid (5 mM, Sigma-Aldrich, St Louis, MO, n = 10), MCT1/2 inhibitor AR-C155858 (100 nM, Adooq, n = 10), 3-mercaptopicolinic acid (100 µM, 3MPA, Toronto Research Chemicals, n = 8), or vehicle (MilliQ water, n = 8). We use a glass micropipette adapted to a Hamilton syringe, this procedure is minimally invasive and there was no mortality associated. Tissues for immunohistochemistry analysis were obtained at P3, by killing the animals by anesthetic overdose followed by a transcardial perfusion with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.3. The mouse brains were post-fixed for 8–12 h, cryoprotected, and kept frozen. Coronal sections of 40 μm thickness were collected in a cryoprotective solution and stored at −30°C until further use.
BrdU Labeling
Thymidine analog 5-bromo-2-deoxyuridine (BrdU; Sigma-Aldrich) was injected intraperitoneally (5 mg/10 g body weight) 3 h after lactate or vehicle injection to P0 animals. At P3, animals were perfused and processed as described above. BrdU incorporation was analyzed by immunohistochemistry (rat-anti-BrdU, 1:1000; Abcam, UK). The cell types that incorporated BrdU and their progeny were identified by double immunohistochemistry with antibodies against BrdU and Ki67, Sox2, Tbr2, DCX, or BLbP.
Cell Culture
Neurons were obtained from embryonic brains as described elsewhere (Ortega and Alcantara 2010). Briefly, a time-pregnant mouse was killed by cervical dislocation and the embryos extracted at embryonic day 16 (E16).
Cerebral cortices were dissected free of the meninges in a solution of PBS with 0.6% glucose (Sigma-Aldrich) and 0.3% bovine serum albumin (Sigma-Aldrich) and then digested with trypsin (Biological Industries, Israel) and DNAse I (Sigma-Aldrich) for 10 min at 37°C. The tissue was mechanically dissociated, centrifuged, and resuspended in CO2-equilibrated Dulbecco's modified Eagle's medium supplemented with 10% normal horse serum (NHS), 1% pen-strep, 0.5 mM l-glutamine, and 5.8 µL NaHCO3 (Sigma-Aldrich)/mL. The cell suspension was preplated at 37°C. After 30 min, the supernatant was collected, centrifuged (1000 g for 5 min), resuspended in NB neuronal culture medium (1% NHS, 1% pen-strep, 0.5 mM l-glutamine, 22 μM glutamic acid (Sigma-Aldrich), 2% B27 (Gibco, USA), and 5.8 µL NaHCO3/mL), and plated at a density of 2.5 × 105 cells/cm2 directly on tissue culture plates coated with poly-d-lysine (Sigma-Aldrich). After 24 h, the medium was replaced with serum-free neuronal culture medium (1% pen-strep, 0.5 mM l-glutamine, 2% B27, 5.8 µL NaHCO3/mL) during 1 days in vitro (div), 3 div, and 5 div. Under these conditions, we obtained a neuron-enriched culture in which about 10% of the cells were glial cells.
The samples were fixed in 4% PFA for 15 min at room temperature (RT) for immunofluorescence, protein extraction and western blot analysis, and ATP measurements. Media were collected to measure lactate and glucose concentrations.
Western Blot/Immunofluorescence
For western blot analysis, protein extracts were obtained from primary cultures. Total cell proteins were separated by SDS-PAGE and electrotransferred from the gel to a nitrocellulose membrane (Bio-Rad). The membranes were blocked and then incubated first with primary antibodies overnight at 4°C, and then with their corresponding secondary HRP-conjugated antibodies (1:3000; Santa Cruz Biotechnology, USA). Protein signals were detected using the ECL chemiluminescent system (Amersham, GE Healthcare, PA, USA). Densitometry analysis, standardized to F-actin as a control for protein loading, was performed using ImageJ software. For quantification, triplicate samples were analyzed and at least 2 different experiments were conducted.
For immunofluorescence, fixed samples (4% PFA for 15 min at RT) were incubated with primary antibodies and appropriate Alexa 488 or Alexa 555 secondary antibodies (1:500, Molecular Probes, USA). The following primary antibodies were used: goat anti-actin (cytoskeleton marker, 1:2000, Santa Cruz Biotechnology), mouse anti-Tuj-1 (neuronal marker 1:10000, Covance), rabbit anti-Tbr2 (neuronal restricted progenitor cells marker, 1:500, Abcam), rabbit anti-Ki67 (proliferation marker, 1:500, Abcam), rabbit anti-Sox2 (NSC/nonrestricted progenitor marker, 1:1000, Abcam) goat anti-MCT2 (neuronal monocarboxylate transporter marker 1:500, Santa Cruz Biotechnology), rabbit anti-PEPCK-M (mitochondrial phosphoenolpyruvate carboxykinase, 1:1000, Abcam), goat anti-GPR81 (G-protein-coupled receptor 1:500, Santa Cruz Biotechnology), rabbit anti-phospho-AMPKα (adenosine monophosphate-activated protein kinase marker 1:1000, Cell Signaling), rabbit anti-Sirt1 (NAD-dependent deacetylase sirtuin-1 marker 1:1000, Upstate), rabbit anti-phospho-PDH-E1α (phosphorylated pyruvate dehydrogenase E1-alpha, 1:5000, Millipore), rat-anti-BrdU (cycling cells marker, 1:1000, Abcam), rabbit anti-DCX (doublecortin, immature neuronal marker, 1:6000, Abcam), rabbit anti-BLbP (glial marker, 1:3000, Chemicon) rabbit anti-AKT (serine/threonine-specific protein kinase, 1:1000, Millipore), rabbit anti-laminin-EHS (1:500, Sigma-Aldrich, S9393), rabbit anti-caspase-3 (apoptosis marker 1:100, Santa Cruz Biotechnology), MitoTracker (250 nM, mitochondria marker, Invitrogen), and To-Pro-3 iodide (Topro) (nuclear stain, 1:500, Molecular Probes). Finally, the preparations were coverslipped with Mowiol (Calbiochem) for imaging.
Lactate Treatments
E16 primary neuronal cultures treated with l- or d-lactic acid (Sigma-Aldrich) were cultured in Neurobasal medium supplemented with 25 mM glucose (glucose medium) or with 25 mM glucose and 4 mM lactic acid ((glucose + lactate medium), or with 4 mM lactic acid only (glucose-free lactate medium). Neurons were cultured for 24 h in the above media supplemented with 1% NHS, 1% pen-strep, 0.5 mM l-glutamine, 22 μM glutamic acid, 1× B27, and 5.8 μL NaHCO3/mL. The medium was then replaced with the same medium but serum-free and the cultures incubated for 4 more days.
In some experiments, lactate or glucose medium was replaced after 5 div with glucose or lactate medium for 1 h (short incubation) or 2 days (long incubation). All experiments were carried out at least 3 times and triplicate samples from each were analyzed by western blot, Inmunofluorescence (as a measure of cell death), or spectrophotometrically (to determine the lactate content of the medium).
ATP Measurements
ATP in cell extracts was measured using a luciferin/luciferase assay (BioVision). Cell extracts were prepared in a single-step boiling water procedure (Yang et al. 2002). In brief, the cells were washed twice with PBS and then suspended in 200 µL of boiling water. The cell suspension was centrifuged (12000g, 5 min, 4°C) and the resulting supernatant was assayed. Ten µL of the cell suspension was mixed with 10 µL of luciferin/luciferase reagent and 80 µL of deionized water. Light emission was measured on a TD 20/20 luminometer (Turner Designs, Houston, USA). A standard ATP curve was obtained by serial dilutions of 10 mM ATP (Sigma-Aldrich). The amount of ATP in the cell extracts was normalized to the cellular protein concentration.
Oxidative Stress
Oxidative stress was measured using the cell-permeable fluorogenic probe CellROX Green (Invitrogen) as recommended by the manufacturer. In its reduced state CellROX Green is weakly fluorescent whereas upon oxidation by reactive oxygen species it binds to DNA and exhibits bright green fluorescence (excitation/emission: 485/520 nm). Cells assayed by this method were cultured in lactate medium as described above; after 5 div they were shifted to glucose or lactate medium for either 1 h (short incubation) or 2 days (long incubation). The CellROX Green reagent was added at final concentration of 5 µM and the cultures were incubated at 37°C for 30 min. The cells were fixed with 4% PFA, mounted with Mowiol, and their fluorescence was then analyzed using ImageJ software (National Institutes of Health, USA).
Experiments testing the effects of MCT2 inhibition or the lactate receptor agonist GPR81 were carried out as follows: E16 primary neuronal cultures were treated with l- or d-lactic acid (Sigma-Aldrich) and cultured in 3 different media: NB medium (with 25 mM glucose, glucose medium), NB medium supplemented with 4 mM lactic acid (glucose + lactate medium) or glucose-free NeurobasalA medium (NBA) supplemented with 4 mM lactic acid (lactate medium). Neurons were cultured for 24 h in these media, also containing 1% NHS, 1% pen-strep, 0.5 mM l-glutamine, 22 μM glutamic acid, 1× B27, and 5.8 μL NaHCO3/mL. The medium was then changed to serum-free formulations containing 1% pen-strep, 0.5 mM l-glutamine, 1× B27, 5.8 μL NaHCO3/mL and the presence or absence of the MCT2 inhibitor AR-C155858 (100 nM, Adooq) or the GPR81 agonist, 3,5-dihydroxybenzoic acid (1 mM, 3,5-DHBA, Santa Cruz) for 4 more days.
In experiments testing the effects of PEPCK inhibition, the cells were cultured as described above, except that the serum-free medium contained either DMSO (1:2000, Sigma-Aldrich) for control conditions or the PEPCK inhibitor 3-mercaptopicolinic acid (100 µM, 3MPA, Toronto Research Chemicals). The cells were then cultured for 4 more days.
l-Lactate Assay
The concentration of l-lactate was determined using an enzymatic reaction based on the oxidation of l-lactate to pyruvate by lactate dehydrogenase (5 mg of the enzyme (Roche)/mL, 550 U/mg) in the presence of NAD (Sigma-Aldrich). In this assay, the amount of NADH produced in the reaction is proportional to the amount of l-lactate in the samples. With this enzymatic system, d-lactate is not detected.
All experiments were carried out at least 3 times and in triplicate samples. The latter were diluted 1:20 with reaction mix [0.3 M hydrazine sulfate (Merck) and 0.87 M glycine (AppliChem), pH 9.5; 2.5 M NAD+ (Sigma-Aldrich), 0.19 M EDTA (Merck)]. Lactate dehydrogenase was added at a final concentration of 6.9 U/mL. The NADH concentration was determined by using the Fluostar Optima BMG Labtech system to measure absorbance (340 nm) and fluorescence (excitation 340 nm/emission 460 nm) 0 and 20 min after the start of the reaction. The endpoint of the reaction was set at 20 min and the corresponding values were used in the calculations. Different concentrations of sodium l-lactate (Sigma-Aldrich) served as the standard.
Imaging and Cell Analysis
Digital images were taken throughout the study using a software-controlled digital camera. Fluorescent preparations were visualized and micrographs were captured with either a Leica TCS-SL Spectral confocal microscope (Leica Microsystems, Mannheim, Germany) or a Nikon Eclipse 800 light microscope (Nikon, Tokyo, Japan). The images were assembled in Adobe Photoshop (v. 7.0), with identical adjustments for contrast, brightness, and color balance to obtain optimum visual reproductions of the data. Morphometric and quantitative analyses were performed using ImageJ software (National Institutes of Health, USA).
Statistical Analysis
Cell counts were expressed as mean cells/mm2 ± standard deviation. The values are the average of 3 replicates of at least 2 different experiments. Statistical analysis was performed using the Statgraphic-plus software and GraphPad Prism. One-way ANOVA and Fisher's least significant difference (LSD) procedure were used to discriminate between the means.
Effect of Lactate on Neuronal Progenitor Survival and Differentiation
To explore the influence of l-lactate on the self-renewal and differentiation of neuronal progenitors in vitro, primary embryonic neuronal cultures were grown in serum-free culture medium with glucose (25 mM glucose, glucose medium), glucose and l-lactate (25 mM glucose + 4 mM l-lactic acid, glucose + lactate medium), or l-lactate only (4 mM l-lactic acid, lactate medium) (see Material and Methods). Under these 3 conditions, initial cell adherence was the same while in the presence of lactate, glial content was residual or mostly absent (Alvarez et al. 2013). After 1 day in vitro (1 div) (Fig. 1A) similar counts were obtained for both the total number of cells (glucose: 274 ± 43, glucose + lactate: 243 ± 46, and lactate: 246 ± 36) and the number of Ki67+ cycling progenitors (glucose: 38 ± 9, glucose + lactate: 44 ± 14, and lactate: 55 ± 14) (Fig. 1C). After 5 div, the neuronal cultures showed signs of differentiation, and neurons identified by staining with the neuronal marker Tuj-1 exhibited well-developed neurites, particularly in glucose medium (Fig. 1B). At this time point, the total cell numbers in the glucose (375 ± 47, P < 0.01) and glucose + lactate (297 ± 40, P < 0.05) cultures were significantly higher than at 1 div while in lactate medium there was no change (247 ± 27). A similar comparison of Ki67+ progenitors (Fig. 1D) showed fewer numbers of these cells in glucose medium (4 ± 3, P < 0.01), while the number remained constant in glucose + lactate medium (57 ± 19) and had significantly increased in lactate medium (79 ± 18, P < 0.01). As post-mitotic neurons cannot reenter the cell cycle, the increase in cell number in 5 div embryonic neuronal cultures can be explained by the division of preexisting Ki67+ progenitors at 1 div. Therefore, the increase in cell number and the depletion of progenitors after 5 div in glucose medium is compatible with 2 rounds of progenitor division; first an asymmetric division giving rise to a progenitor and a differentiated cell, and then a symmetric division of the progenitor giving 2 differentiated cells. The observation in glucose + lactate medium of an increased number of cells together with progenitor maintenance is compatible with one round of asymmetric progenitor division giving rise to a self-renewing progenitor and a differentiated cell; and finally, the increase in progenitors but not in the total cell number in lactate medium suggests self-renewing symmetric divisions and an enrichment in progenitor cells compared with the initial pool of terminally differentiated cells.
Based on these observations, we speculated that glucose promotes the differentiation of neuronal progenitors while lactate is required for their self-renewal. To test this hypothesis, neuronal cultures were grown in lactate medium for 5 div and then for 2 more days either in the same medium or in glucose medium (Supplementary Fig. 1A). After the 7 days, there were significantly fewer Ki67+ progenitors in glucose than in lactate medium (10 ± 1 and 63 ± 5 respectively, P < 0.001, Supplementary Fig. 1B), while the total number of cells was significantly higher in glucose than in lactate cultures (220 ± 41 and 170 ± 23 respectively, P < 0.001, Supplementary Fig. 1C). These results suggested that glucose medium induced one terminal division of Ki67 progenitors whereas in lactate medium these cells remained mostly quiescent. When the opposite experiment was done, i.e., neuronal cultures were grown in glucose medium for 5 div and then for 2 more days either in the same medium or in lactate medium (Supplementary Fig. 1D), there were no changes either in the total number of cells (260 ± 34 glucose; 228 ± 44 lactate) or in the number of Ki67+ progenitors (2 ± 2 glucose; 1 ± 1 lactate) (Supplementary Fig. 1E,F), indicating that lactate cannot reprogram the terminal progenitor differentiation induced by glucose.
We then examined whether lactate induced the formation of reactive oxygen species, leading to oxidative stress and cell death and therefore the observed differences in cell number. Neuronal cultures were grown in lactate medium for 5 div and then in the same medium or in glucose medium. After either 1 h or 2 days, the cells were treated with CellROX, an indicator of oxidative stress, for 30 min before fixation. After 1 h, CellROX fluorescence was higher in neuronal cultures grown in glucose than in lactate medium (21 ± 3 and 16 ± 2 cells respectively, P < 0.001). After 2 days, however, CellROX intensity decreased and the differences disappeared (glucose: 10 ± 1 cells, lactate: 10 ± 5 cells) (Supplementary Fig. 1G,H). As an indicator of cell death, the number of caspase-3+ cells was analyzed under the same experimental conditions. The percentage of dead cells was very low in either medium although at 1 h it was slightly higher in the lactate than in the glucose-containing cultures (glucose: 2 ± 2, lactate: 5 ± 2, P < 0.05). These differences were no longer observed after 2 div (glucose: 3 ± 3, lactate: 2 ± 3) (Supplementary Fig. 1I,J).
Effect of Lactate on Lactate-signaling and Metabolism
Next, we analyzed whether the presence of lactate in the medium affected the expression of lactate-related machinery, i.e., the high-affinity proton-linked monocarboxylate transporter MCT2 (Halestrap and Wilson 2012), the G-protein-coupled lactate receptor, GPR81 (Lauritzen et al. 2013), and the mitochondrial enzyme PEPCK-M, required for the anabolic use of lactate in the liver (Mendez-Lucas et al. 2013). After 5 div in glucose medium, MCT2 was detected at low levels in neuronal membranes. In lactate media MCT2 was abundantly expressed in membranes and cytosol of neurons, Ki67+ and Tbr2+ progenitors (Fig. 2A,B). Tbr2 transcription factor is required for the conversion of radial glia into neuronal restricted progenitors (Sessa et al. 2008), that is the principal type of progenitor induced by lactate treatments (Alvarez et al. 2013). In contrast, GPR81 (Fig. 2C) was expressed in neuronal membranes in glucose medium and by some but not all Ki67+ progenitors (not shown) and Tbr2+ neuronal progenitors in lactate conditions. Similarly, in glucose medium, PEPCK-M was detected at low levels, with a punctate distribution in the neuronal soma, whereas in the presence of lactate both neurons and progenitors expressed very high levels of the enzyme (Fig. 2D). Quantitative analysis by western blot and densitometry (Fig. 2E,F) corroborated the significant lactate-related increases of these 4 proteins, MCT2, GPR81 and PEPCK-M, and Tbr2. Moreover, PEPCK-M and Tbr2 expression was significantly higher in lactate than in glucose + lactate medium. The absence of Sox2+ progenitors (Fig. 2E,F) and astrocytes (Alvarez et al. 2013) in lactate cultures suggested that the Ki67+ progenitors were mostly Tbr2+ neuronal progenitors and that lactate and PEPCK-M activity allowed the avoidance of differentiation, perhaps by promoting the maintenance of self-renewal.
In a pharmacological approach we sought to answer the question whether neuronal survival and progenitor maintenance required lactate intake, either through monocarboxylate transporters, activation of the GPR81 lactate receptor, PEPCK-M catalytic activity, or a combination of all 3. Thus, neuronal cultures were grown for 5 div in the presence or absence of 100 nM of the MCT1/2 inhibitor AR-C155858 beginning at 1 div. When added to glucose + lactate medium, the inhibitor induced Ki67+ progenitor depletion (22 ± 5 and 0 ± 1 respectively, P < 0,001), without affecting neuronal survival (211 ± 51 and 240 ± 24, respectively), whereas when added to lactate medium it induced massive cell death (159 ± 35 and 0, respectively) (Fig. 3A). In a second set of experiments, 3,5-DHBA, a selective agonist of GPR81, was added to glucose medium or to glucose/lactate-free medium beginning at 1 div. After 5 div in glucose medium, the agonist did not modify either the total number of cells (glucose: 255 ± 46, glucose + agonist: 216 ± 79) or the number of Ki67+ progenitors (glucose: 0 ± 0, glucose + agonist: 0 ± 0), nor did it promote cell survival in glucose/lactate-free medium (Fig. 3B). Moreover, the effects of l-lactate on neuronal cultures were not mimicked by d-lactate (Supplementary Fig. 2).
To specifically examine the potential role of PEPCK in shuttling carbons from lactate through cataplerosis into various metabolic pools, we treated cultures during 5 div with 3-mercaptopicolinic acid (3MPA), a well-known inhibitor of PEPCK activity, or with DMSO as the control (Fig. 3C,D). In glucose medium, PEPCK inhibition did not affect the total number of cells (256 ± 50 DMSO; 252 ± 54 3MPA) or the number of Ki67+ progenitors (1 ± 2 DMSO; 1 ± 2 3MPA). However, in glucose + lactate or lactate medium, the lack of PEPCK activity corresponded with a significant reduction in the total number of cells (from 204 ± 47 to 150 ± 46 in glucose + lactate and from 121 ± 50 to 79 ± 34 in lactate, P < 0.01) as well as the complete depletion of Ki67+ progenitors (from 12 ± 5 glucose + lactate, 15 ± 6 lactate to 0, P < 0.01), indicating that in the presence of lactate, PEPCK-M inhibition leads to progenitor cell death. Consistent with this finding, lactate consumption decreased in the presence of 3MPA (from 0.7 ± 0.1 to 0.2 mM in glucose + lactate, and from1.7 ± 0.1 to 1.5 ± 0.2 mM in lactate, P < 0.05) (Fig. 3E).
Taken together these data suggest stereospecific effects of l-lactate on neuronal survival and progenitor self-renewal. These effects are apparently mediated by the cellular entry of l-lactate through MCT1/2 receptors and its metabolism at the TCA junction, represented by PEPCK-M catalytic activity.
Lactate-associated Changes in the Metabolic Profile of Neuroprogenitor Cells
To investigate changes in the metabolic profile of self-renewing progenitor cells in the presence of lactate several key elements of metabolic regulation were analyzed. Differences in mitochondrial distribution were visualized by epifluorescence using MitoTracker, a cell-permeable mitochondrial marker. In neuronal cultures grown in glucose, glucose + lactate, or lactate medium for 5 div well-developed neurons stained with the neuronal marker Tuj-1 contained fewer mitochondria than young neurons and progenitors (Tuj-1 negative), which showed intense mitochondrial labeling (Fig. 4A). Consistent with progenitor enrichment, mitochondrial staining was highest in lactate medium, while there was no medium-related difference in ATP levels (Fig. 4B). Western blot and densitometry analysis showed slightly higher levels of phosphorylated AMPK (AMPK-P) in glucose + lactate medium, without concomitant changes in NAD-dependent deacetylase sirtuin-1 (Sirt1) or phosphopyruvate dehydrogenase (PDH)-E1−α [PDH E1α-P(Ser293)], whereas AMPK-P, Sirt1, and phospho-PDH-E1−α levels were much lower in lactate condition (Fig. 4C,D) than in either of the glucose-containing media, indicating striking increased in the energy charge, redox potential, and mitochondrial metabolism of lactate through PDH. These data are consistent with a dramatic shift to oxidative metabolism as lactate becomes the main energy and carbon source in the cell. The metabolic profile described is a newly identified hallmark of neuron restricted progenitor cells and contrasts with the glycolytic profile of glucose restricted cells (mainly the glial component).
Lactate restriction of neuronal progenitors implies that lactate metabolism also plays a crucial role in sustaining the increase in biomass that occurs during the cell cycle and in the ECM production characteristic of these cells. In this context, PEPCK-M is the only pathway that can export carbons from lactate and other TCA cycle intermediates (i.e., glutamine) into the triose and hexose pools, through PEP synthesis (Mendez-Lucas et al. 2013). Therefore, we evaluated whether the presence of lactate and the inhibition of PEPCK-M activity using 3MPA altered the synthesis of laminin, a highly glycosylated ECM protein that serves as a marker of synthetic activity in neuronal progenitors (Kazanis et al. 2010). Indeed, lactate significantly increased the laminin content of neuronal cultures (Fig. 4E,F), mainly associated with progenitor cells (Supplementary Fig. 3). In the presence of lactate and 3MPA there was a substantial decrease in the laminin content as well as in Tbr2+ neuronal progenitors as shown by western blot (Fig. 4G,H). This result suggested that the PEPCK-M pathway is relevant to link the mitochondrial and glycolytic pools of building blocks necessary to sustain biosynthetic processes.
Requirement of Lactate for Neural Progenitors in vivo
We tested whether progenitor maintenance in the cerebral cortex was effectively mediated by l-lactate and PEPCK-M catalytic activity. In this in vivo experiment, 2 µL of either l-lactate (5 mM, lactate), the PEPCK-M inhibitor 3MPA (100 µM, 3MPA), MCT1/2 inhibitor AR-C155858 (100 nM, MCT inhib), or vehicle (control) was injected into the lateral ventricle of newborn (postnatal day 0, P0) mice (Fig. 5A). The effects on PEPCK-M expression and in the number of Ki67+ cycling cells, Sox2+ NSC/nonrestricted progenitors, and Tbr2+ neuronal progenitors were analyzed in the ventricular/subventricular and intermediate zones (VZ/SVZ, IZ) at P3 (Fig. 5). The results showed the PEPCK-M expression in the germinal VZ/SVZ of lactate-injected brains (Fig. 5B) together with significant increases in the number of Ki67+ progenitors (vehicle: 83 ± 14, lactate: 129 ± 37, MCT inhib: 73 ± 11, 3MPA: 45 ± 13; Fig. 5C,D), Sox2+ NSC/progenitors (vehicle: 94 ± 13, lactate: 125 ± 33, MCT inhib: 80 ± 22, 3MPA: 57 ± 9; Fig. 5E,F), and Tbr2+ neuronal progenitors (vehicle: 57 ± 10, lactate: 72 ± 12, MCT inhib: 34 ± 12, 3MPA: 23 ± 8; Fig. 5G,H). Conversely, the numbers of all these cells decreased significantly in 3MPA while only the number of Tbr2+ neuronal progenitors was significantly reduced by the MCT inhibitor. Moreover, compared with the vehicle control, laminin expression in the germinal VZ/SVZ dramatically increased in lactate-injected animals but decreased in 3MPA and MCT inhibitor-injected animals (Fig. 5I). Taken together these data provide in vivo corroboration of the lactate dependence of neural progenitors.
Finally, we examined lactate-mediated changes in progenitor fate using BrdU, a marker for DNA replication. BrdU was injected 3 h after lactate or vehicle (P0) administration and incorporation into different cell types was analyzed at P3 (Fig. 6A). The total number of BrdU labeled cells was significantly higher in lactate injected animals than in controls (control: 98 ± 31, lactate: 122 ± 38, P < 0.05). In lactate injected animals there was a significant increase in the number of BrdU+ progenitors expressing Ki67 (vehicle: 39 ± 11, lactate: 54 ± 12; Fig. 6B,C), Sox2 (vehicle: 27 ± 11, lactate: 38 ± 7; Fig. 6D,E), and Tbr2 (vehicle: 4 ± 1, lactate: 7 ± 1,; Fig. 6F,G). However, the number of double labeled BrdU and DCX+ immature neurons (vehicle: 18 ± 7, lactate: 15 ± 3; Fig. 6H,I) or BLbP+ glial cells (vehicle: 11 ± 3, lactate: 12 ± 4,; Fig. 6J,K) were similar in both treatments. The consequence is a net increase in the total number of progenitors 3 days after lactate injection without significant changes in the number of differentiated neurons and glial cells generated in this period. These data might indicate that neonatal lactate maintains progenitor self-renewal in vivo, corroborating in vitro results.
Metabolism is an important indicator of cell function, since it shifts together with differentiation, growth, or anabolic capacities. For example, undifferentiated stem cells preferentially rely on glycolysis whereas differentiated cells up-regulate oxidative metabolism to support their anabolic or biological potential (Facucho-Oliveira and St John 2009; Ivanovic 2009; Simsek et al. 2010). In the developing brain, NSC/radial glia progenitors generate the various differentiated cell types (Rowitch and Kriegstein 2010). Several studies have shown that the highly hypoxic environment characteristic of the stem cell and NSC niche favors glycolysis and lactate production at sites of unrestricted progenitor cell proliferation (Mohyeldin et al. 2010). As NSCs mature, their specific lineage restriction is likely to be accompanied by a shift in their metabolic needs (Stubbs et al. 2009; De Filippis and Delia 2011; Goldman and Chen 2011). This shift might explain the role of l-lactate, previously shown to be a critical substrate of the developing brain (Medina and Tabernero 2005; Rinholm et al. 2011). Indeed, our results provide evidence of a lactate-restricted metabolic requirement to fulfill the biosynthetic needs of neuronal restricted progenitors (summarized in Fig. 7).
Aerobic glycolysis is a functional marker of the specialized phenotype of dividing cells, whether they arise from physiologic tissue components (i.e., stem cells or endothelial cells) or after pathologic dedifferentiation (tumor cells) (De Bock et al. 2013; Suda et al. 2011; Takubo et al. 2013). This seemingly inefficient, partial oxidation of glucose nonetheless provides both a rapid source of energy and the anabolic building blocks to support cell division, but it reduces the cellular capacity to integrate into a functional, complex tissue. The same can be said for NSC metabolism (Yamasaki et al. 2001; Rafalski and Brunet 2011) and of radial glia progenitors and astrocytes, which are also essentially glycolytic (Tsacopoulos and Magistretti 1996; Yamasaki et al. 2001). In contrast, in this work we showed that neuronal restricted progenitors have an oxidative metabolic profile that is entirely dependent on lactate as a carbon source. In the presence of glucose, lactate withdrawal induces progenitor differentiation to a neuronal fate.
Consistently, key metabolic markers of oxidative metabolism, including increased mitochondrial content and high PDH activity (reduced phosphorylation), are classically found in differentiated cells with a high anabolic capacity, such as liver, heart and slow-twitch muscle, and pancreatic β-cells. Terminal oxidative phosphorylation of pyruvate, whether from glucose or lactate sources, ensures the appropriate redox potential and energy charge needed to sustain biosynthesis at a high level of efficiency; while ensuring minimal rates of cell division. The progenitors examined in this study underwent one or 2 rounds of cell division during their 5 days in culture, even though in all cases their nuclei were Ki67+. The cellular redox state contributes to the determination of cell fate. Thus, for example, increases in Sirt1 activity under oxidizing conditions induce neural progenitors to adopt a glial fate (Prozorovski et al. 2008), while under basal nonoxidizing conditions these progenitors differentiate along a neuronal line (Hisahara et al. 2008). Our data showed low-level Sirt1 expression, consistent with an increased redox potential, in parallel with a restriction to a neuronal progenitor fate. Also, diminished AMP-K phosphorylation suggested that when lactate is the main carbon source both redox potential and energy charges are increased, in line with the requirements of a mainly anabolic metabolism. Hence, we propose that Tbr2+ neuronal restricted progenitors have a role in maintaining the ECM of the neurogenic niche and in the secretion of factors necessary for the neurogenic environment. Laminin is an integral component of the ECM of neurogenic niches (Kazanis et al. 2010) and, as shown in the present work and in a previous study (Alvarez et al. 2014), it is secreted by neural cells and progenitors exposed to lactate. Indeed, we found that laminin production was sensitive to the PEPCK inhibitor 3MPA, suggesting that both PEP and TCA flux are necessary to support biosynthetic processes, such as laminin production and glycosylation. PEPCK activity is present in the cytosol (PEPCK-C) and in mitochondria (PEPCK-M), encoded by 2 different nuclear genes, PCK1 and PCK2, respectively. In neural progenitors, only the mitochondrial isoform, PEPCK-M, was detected, with no measurable cytosolic protein found (data not shown). PEPCK-M catalyzes the GTP-dependent decarboxylation of mitochondrial oxaloacetate to produce PEP, which is then exported into the cytosol where it feeds the reverse glycolytic pathway (Stark et al. 2009; Mendez-Lucas et al. 2013). PEPCK-M has important advantages over PEPCK-C, since the shuttling of malate is not required and enzyme activity is coupled to TCA cycle flux through the recycling of GTP produced in the succinyl-CoA synthase reaction (Stark et al. 2009). This pathway also shuttles carbons from lactate into the triose-phosphate intermediate pool for the synthesis of serine/glycine or glycerol-3P, or into hexose-phosphate intermediates for protein glycosylation, in the absence of oxidative phosphorylation (Nye et al. 2008; Yang et al. 2009; Kalhan and Hanson 2012). It is noteworthy that PEPCK-M is the only known pathway that can communicate mitochondrial carbon intermediates directly to the glycolytic pool, because of the irreversible nature of the pyruvate kinase step.
Lactate injection in vivo produced similar results, and confirmed that lactate increased the progenitor BrdU-traced pool. As described in vitro, lactate induced PEPCK-M expression, laminin synthesis and Tbr2+ neuronal restricted progenitors in vivo, and the inhibition of lactate machinery (MCT1/2 lactate transporter or PEPCK-M activity) significantly reduced the number of neuronal restricted progenitors. In vitro, Sox2+ cells with NSC characteristics were rarely seen in our neuronal cultures, however in vivo lactate injection induced an increase in Sox2+ progenitors and the inhibition of PEPCK-M activity significantly reduced their number. These data are in accordance with previous results demonstrating the requirement of the NSC niche for specific topographical cues. Thus, while Tbr2+ progenitors are induced by a flat substrate of lactate-releasing polylactic acid (PLA), Sox2+ progenitors/NSC are only induced when the same PLA substrate is molded as aligned nanofibers, a 3D organization that reproduces the aligned palisade of embryonic radial glia (Alvarez et al. 2013; 2014).
On the other hand, MCT1/2 inhibitor in vivo only affected the number of neuronal progenitors, as compared with the broad progenitor reduction observed after PEPCK-M activity blockage. The complexity of lactate transport across membranes in vivo might explain these data as it is dependent on MCT1-4 transporters expression and modulated by intra and extracellular concentration, pH and proton gradient, and in addition lactate can enter the cell trough other ways including gap junctions (Hertz et al. 2014). MCT2 is expressed in the neuronal lineage while MCT1 and 4 are found in glial cells (Debernardi et al. 2003).
Taken together, our results support the strict dependence of neuronal restricted progenitors on lactate metabolism and on the provision of ECM components of the neurogenic niche by the anabolic activity of PEPCK-M. In the in vivo niche, other physical and/or biochemical cues might cooperate with lactate to maintain different progenitor populations. Further experiments will be required to determine the impact of lactate metabolism modulation on the neuronal composition and functional organization of the cerebral cortex.
Supplementary Material
Supplementary material can be found at: http://www.cercor.oxfordjournals.org/
This study was supported in part by grants from Spain's Ministerio de Economía y Competitividad (MAT2011-29778-C02-02) and (BFU2012-37177), cofinanced by the European Regional Development Fund to S.A and J.C.P respectively; and from Generalitat de Catalunya 2009 SGR719 to S.A.
Z.A acknowledges the fellowship from IBEC 10-2009-01 and P.H was supported by a fellowship from the Ministerio de Educación y Ciencia (FPU). We are grateful to Wendy Ran for editorial assistance, to J.A. Ortega for the drawing in Figure 5A and B. Torrejón from the Scientific-Technical Services UB (Campus Bellvitge) for technical support in confocal microscopy. Conflict of Interest: None declared.


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Hi everyone! I am new to forums and thought i would post my story to see if anyone was in a similar situation to myself!
I am 24 and my OH and I have decided to TTC.
I have recently stopped the pill and am on my first natural cycle with 7 days to OV. We stated trying last month but turns out going off the pill 'readjusted' my cycle so we have started again.
The catch is my OH leaves for a six month work trip in NOV so we have limited opportunity to try. Has anyone got any advice? I have been doing alot of reading on fertiliy and conception but i wouldnt mind some advice or tips from women who have been trying. We are so excited but i know i will be devostated if it doesnt happen before his trip..Most of all as we will not be able to try again until Dec next year when I get home from a year of work trips.


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September 8th, 2016
Enter for a chance to win a $5000 Willowbrook gift card (must be able to pick up the prize in person in Langley, BC). Good luck!
Expires October 16, 2016
Entry Frequency: single
Not open to: QC
Age: 18+
One Response to Win a $5000 Willowbrook Gift Card
Brittaney Gosselin says:
This would be handy
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Your fundraising step by step
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Decide how much money you want to raise and let everyone know.
We can provide you with Ovarian Cancer Action branded boxes or tin to collect funds at your event. If you are planning a large event we also have buckets available.
When you have finished your collection, please return all tins or buckets to us as they can be used by other supporters, which helps to save us money.
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Let your employer know about the fantastic event that you're planning and they may be generous enough to offer matched giving, for every pound you raise – they might donate a pound too.
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Try to keep your costs as low as possible, do you know anyone that might be willing to help you out for free?
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- Create a page on BT my donate. We encourage our supporters to use My Donate because of their low administration costs, meaning more of your money goes towards our research. However, we also accept donations through Just Giving, Virgin Money Giving and any other online fundraising system.
- Download a sponsorship form and pay in using credit or debit card. To submit your money via credit or debit card call us on 0207 380 1730 or visit www.justgiving.com/oca
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This image is one of my 'primitive art" series.
birds cat cats kiss kitten kitty love mariage nature relationships sky
What a beautiful drawing! Well done! (Thanks for your comment!)
Wonderful art work…lots of talent!
Thank you kari and Elen
very unique style! this is lovely and very sweet :)
ochen milaja kartinka :
Your work is beautiful :) I love your style!
Thank you , guys!
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- Details
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text: I qualify as an ODP (Operating Department Practitioner) in September this year and I've been seriously thinking about joining my local TA RAMC Field Hospital in Nottingham. The problem I have is that I'm 39. Although they say you can join up to the age of 43 do many people my age actually join up or will I look like a big div doing TA selection alongside 18 year olds? I keep myself fairly fit - run 1.5 miles in 09.32, 82 press ups/78 sit ups in two minutes - so I think I meet the minimum standard there and I think my ODP qualification will be of interest to the TA. Also, what's the 'retirement age' for the TA? Thanks Rusty


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Transition Metals
- Created by: Maura1
- Created on: 25-04-14 00:00
First 818 words of the document:
Transition Metals and the d-Block Elements Answers
1. In an atom of a d-block element, electrons go into the 4s subshell before the 3d subshell. When a d-block atom
forms an ion electrons are lost from the 4s subshell first before losing from the 3d subshell. The general
configuration for a d-block element is [Ar] 3dx 4s2, where x is the number of the column along the d-block.
2. In the fourth period, there are two exceptions to the 4s first rule. There is a slight gain in stability in having a full
or half-full set of d-orbitals. Thus chromium has the electron configuration [Ar] 3d5 4s1; copper has the
configuration [Ar] 3d10 4s1.
3. The d-block elements are those between the s-block and the p-block. All d-block elements have an outer
electron configuration of ndx, where x is any number from 1 to 10.
4. A transition metal has one or more unpaired d-electrons in one of its ions. E.g., titanium forms a Ti3+ ion, which has
one unpaired d-electron, and Ti2+, which has two unpaired electrons. Therefore, titanium is a transition metal.
5. Across the third period, the first ionisation energies generally
increase across, due to the increase in nuclear charge, without
an increase in the number of shielding electrons. However, in the
d-block, the outer electron that is removed in the formation of
an ion is a 4s-electron, and it is shielded by the inner
3d-electrons. Although the nuclear charge increases across the
block from scandium to zinc, the number of inner shielding
3d-electrons increases as well. This means that the first
ionisation energies of the d-block elements in period 4 are fairly
6. There is normally a big jump between successive ionisation
energies as a new quantum shell loses an electron. This electron is subjected to a much stronger pull from the
nucleus as it is much less shield. However, the energy of the 3d- and 4s- electrons are very similar in d-block
elements and so the big jump comes after all the 4s- and 3d- electrons have been removed.
7. Metallic bonding can be described as the attraction between the delocalised electrons and the positive ions
formed when the metal atoms lose their valence electrons into the cloud of delocalised electrons. The strength
of the bond determines the melting temperature of the metal. In the d-block metals, both the d-electrons and
s-electrons are used in bonding. Therefore, the melting temperatures of d-block metals are significantly higher
than those of s-block metals. The metals are also much harder and stronger.
8. The metals in the s-block and the non-transition d-block metals exist in only one oxidation state in their
compounds. However, the transition metals exist in different oxidation states, e.g. manganese has stable
compounds such as MnSO4 (+2), MnO2 (+4), K2MnO4 (+6) and KMnO4 (+7).
9. In most compounds in the +2 and +3 oxidation states, the transition metals are ionically bonded, but when the
transition metal is in an oxidation state of +4 or higher, it is covalently bonded, often in an anion. E.g. the
manganese atom in MnO4- ion is covalently bonded to the four oxygen atoms by one single and three double
10. The extra energy required to remove a third electron from an Fe2+ ion to form Fe3+ is small enough that it can be
recovered either from lattice energy or from hydration energy of the cation. However, this is not the case with
calcium, where removal of a third electron would have to be from an inner shell, requiring energy that cannot be
11. When d-block cations are dissolved in water, they become hydrated. The oxygen atom in a water molecule has a
lone pair of electrons that forms a dative covalent bond with an empty 3d- or 4p- orbital in the metal ion. E.g. the
hydrated chromium (III) ion, which has the formula [Cr(H2O)6]3+. Six dative covalent bonds form, so the hydrated
ion has the coordination number 6. This ion is known as a complex ion.
12. The coordination number is the number of near neighbouring atoms that are bonded to the central ion. E.g. In
[Cr(H2O)6]3+, the Cr3+ ion has six oxygen atoms as near neighbours.
13. The shape of a complex ion can be predicted using valence-shell electron-pair repulsion theory. E.g. in
[Cr(H2O)6]3+there are six dative bonds, each containing a pair of electrons. These six pairs of bonding electrons
repel each other to the position of minimum repulsion, which is also the position of maximum separation. The
shape therefore is octahedral.
14. Monodentate ligands use one lone pair of electrons to form a dative covalent bond with the d-block ion:
Other pages in this set
Page 2
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Ammonia, NH3, and organic amines such as ethylamine form complexes. E.g. the complex of Cu2+ and
ammonia, which has the formula [Cu(NH3)4(H2O)2]2+.
Anions such as Cl- and CN- also form complexes, e.g.
[Fe(CN)6]4- is a complex between an Fe2+ ion and six CN- ions.
[CrCl4]- is a complex between a Cr3+ ion and four Cl- ions it is energetically unfavourable to fit six large
ligands around a small cation.…read more
Page 3
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The energy difference between these two sets of d-orbitals in a typical complex ion is equal to the energy
of a photon in the visible region of the spectrum.
When white light shines through a solution of a complex ion of a transition metal, photons of a particular
frequency are absorbed and their energy promotes an electron from the lower energy level to the upper
energy level. This is called a d-d transition.…read more
Page 4
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Chromium is unstable in the +2 state compared with the +3 state. The +2 state can be stabilised by formation of a
complex with ethanoate ions, CH3COO-. The hydrated chromium (II) ions undergo ligand exchange and a
precipitate of red chromium (II) ethanoate complex is formed:
2[Cr(H2O)6]2+ (aq) + 4CH3COO- (aq) [Cr2(CH3COO)4(H2O)2] (s) + 10H2O (l)
36.…read more
Page 5
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E.g. Cu2+ + e- Cu+ E = +0.15 V
45. The feasibility of a redox reaction is indicated by the value of the standard potential of the cell, E cell. Feasibility
is worked out by:
1) Reverse the half equation that has one of the reactants on the RHS and changing the sign of its E value.
2) This half-equation is then added to the half-equation that has the other reactant on the LHS.…read more
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Originally posted on Fox Sports West | Last updated 11/27/12
The Angels have moved to address a weakness that contributed to their falling short of the playoffs, reaching a contract agreement with former Philadelphia Phillies closer Ryan Madson. Madson, 32, will earn a base salary and could make more in incentives, according to two people familiar with the negotiations. The deal is contingent on Madson's passing a physical examination. He has undergone that physical and the results should be available Wednesday, the people said. Madson is a 6-foot-6 right-hander who throws hard and has an excellent changeup. But he is less than eight months removed from Tommy John surgery that kept him sidelined last season. Madson had 32 saves for the Phillies in 2011, signed as a free agent with the Cincinnati Reds, but was injured during training camp. His surgery was performed in April by Dr. Lewis Yocum, the Angels team physician. Madson, who attended Valley View High in Moreno Valley, is represented by agent Scott Boras. Madson has been throwing on flat ground and is expected to resume throwing off a mound in late December or early January. The Angels are hoping Madson will be ready to pitch by spring training. He could be in line to assume closing duties from Ernesto Frieri, who walked 26 batters in 54 13 innings last season and had three of the team's 22 blown saves. Those blown saves were a major factor in the Angels' finishing four games behind the Texas Rangers and Baltimore Orioles for an American League wild-card playoff spot. -Lance Pugmire
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