text:
PEPFAR's annual planning process is done either at the country (COP) or regional level (ROP).
PEPFAR's programs are implemented through implementing partners who apply for funding based on PEPFAR's published Requests for Applications.
Since 2010, PEPFAR COPs have grouped implementing partners according to an organizational type. We have retroactively applied these classifications to earlier years in the database as well.
Also called "Strategic Areas", these are general areas of HIV programming. Each program area has several corresponding budget codes.
Specific areas of HIV programming. Budget Codes are the lowest level of spending data available.
Expenditure Program Areas track general areas of PEPFAR expenditure.
Subdivisions of Program Areas, these track general higher level sub-classifications of expenditure.
Subdivisions of Major categories, these are the most detailed expenditure data.
Cross-cutting attributions are areas of PEPFAR programming that contribute across several program areas. They contain limited indicative information related to aspects such as human resources, health infrastructure, or key populations programming. However, they represent only a small proportion of the total funds that PEPFAR allocates through the COP process. Additionally, they have changed significantly over the years. As such, analysis and interpretation of these data should be approached carefully. Learn more
PEPFAR sets targets using the Monitoring, Evaluation, and Reporting (MER) System - documentation for which can be found on PEPFAR's website at https://www.pepfar.gov/reports/guidance/. As with most data on this website, the targets here have been extracted from the COP documents. Targets are for the fiscal year following each COP year, such that selecting 2016 will access targets for FY2017. This feature is currently experimental and should be used for exploratory purposes only at present.
Years of mechanism: 2013
To increase access to primary health care services in South Sudan, USAID plans to support a five year Service Delivery Project, beginning in April 2012 and ending in April 2017. The goal of the Service Delivery Project is to increase access to quality primary health care services for all people in Central and Western Equatoria States. Through increasing the capacity of health facilities to provide a minimum package of primary health care services and increasing community access to information and services through health education, mobilization, and community based service provision, the Service Delivery Project will support the Ministry of Health in its goal to provide comprehensive health care to all people in South Sudan.
The Service Delivery Project aims to expand access and increase the quality of health services through an integrated, standardized package of services, and increase demand through community mobilization. By increasing access to information and standardized, quality services, the project will ultimately improve health outcomes and reduce morbidity and mortality, especially among women and children under five. HIV prevention is part of the standard package of services, and, where appropriate, includes access to quality PMTCT and testing and counseling services. The objective of the HIV/AIDS program is to develop PMTCT services in selected counties, provide Testing and Counseling through Provider Initiated Counseling and Testing (PITC), facility based Care and Support services as well as TB/HIV referrals
South Sudan depended on Global Fund for the provision of ARVs in the whole Country. Given the fact that there is no treatment for new clients who are eligible for treatment in the next few years, there is need to strengthen care services both at community as well as facility levels. The Service Delivery Project will provide some facility based care services including cotrimoxazole prophylaxis and treatment of OIs. TB/HIV services and referrals will also be provided.
In South Sudan, the human capacity for effective program management is constrained both at the central Ministry and State Ministry of Health. To address this, Jhpiego will participate in and support regular joint support supervision with the state Ministry of Health staffs to improve effectiveness of AIDS programs at the PHCCs. They will also support monthly state HIV/AIDS coordination meetings. At the national level the project will support relevant policy and guideline development through participation in SSAC and MoH National technical working groups for HIV/AIDS, especially PMTCT. Other activities involve support for national activities such as World AIDS Day.
The Service Delivery Project will continue to provide HIV testing and counseling (HTC) for pregnant women at the ANC Clinic as part and parcel of ANC services. PITC services will be coupled with treatment of and/or referrals for treatment of opportunistic infections (OIs). Linkages will be maintained between the facilities and other home and community based psychosocial and economic support.
Other prevention services will be provided through this partner. These services will target vulnerable populations in the project sites both at community and facility level.
The Service Delivery Project will support current PMTCT sites in Western and Central Equatoria States. PMTCT services will be provided to new clients in existing sites but in the next two years there will be no scale up of these services due to limitation of PMTCT funding in South Sudan. The PMTCT implementation utilizes national protocols and guidelines, with the four prongs of Primary prevention of HIV infection among pregnant women: prevention of unintended pregnancies among women living with HIV; prevention of HIV transmission from women living with HIV to their infants; and care, treatment and support for mothers living with HIV, their children and families.
Health workers will be trained in PMTCT provision including the provision of ARVs to pregnant mothers (OGAC is supporting provision of emergency commodities for PMTCT in the next 2 years) on site. Through routine data collection and supervision of the sites, the Service Delivery Project will ensure quality of service and standards are kept. The County Health Department will be involved in monitoring the provision of services at the facilities. The PMTCT sites shall be part of the routine antenatal care services at the sites and integrated with family planning services. Demand creation for services will be part of the community mobilization outreaches from the facilities. There will be continued coordination of activities with partners providing community related HIV/AIDS services.


text:
we also kick players out of our coop's, it's normal that we want to play with our clan. And if some have not yet reached the appropriate level they have to join later.
You certainly have a CLan then play with them, then you can kick out strangers.
I see no problem there

subset: commoncrawl


text:
How to Select The Best Janitorial Services
We all know the importance of having a clean environment and therefore it is important to ensure that we are surrounded with clean carpets and tiles and the only way that we can achieve this is by ensuring that we have reputable cleaning services that will offer good quality services to our offices and homes. In this discussion, we are going to look at how individuals are able to select the best carpet cleaning, tile and grout cleaning and also janitorial services. It is very important to ensure that you factor in the qualification of the specific service provider that will be doing the cleaning services for you and this means that you have to ensure that they have the right certification and are qualified to be able to perform the services at the highest levels. It is important to ensure that the service provider that you decide to select is able to provide a good range of cleaning services so that you can deal with one service provider offering all kinds of cleaning services that you may require as opposed to dealing with numerous service providers to get the various cleaning services that you may want.
It is important to ensure that the service provider is able to provide you with a lasting experience for your carpets and also your tiles so that you can be sure that your surfaces are regularly cleaned at the same time they should be in a position to provide you with discounts for being a regular customer and also good warranty in terms of the services that they provide. The company that you may decide to do or to offer you the cleaning services should be in a position to ensure that they can explain about the kind of chemicals that they use so that you can confirm that they will not have any adverse effects to your employees or your children at home.
The fact that you may be having the most expensive tiles does not mean that they will look attractive forever and therefore you have to ensure that you maintain them well by offering good cleaning services that will leave them to look shiny and attractive at all times. It is normally very important to ensure that you work within your budget or you make sure that you are able to cut down on costs and the only way that you can do this is to do good research so that you can come up with free quotes showing the various charges that you will encounter with the cleaning company so that you may be in a position to settle for a good price that will be within your budget. In this talk, we have looked at the different things that you have to put into consideration when choosing the best services when it comes to carpet cleaning tile and grout cleaning and janitorial services.
Practical and Helpful Tips: Janitors
The Best Advice About Professionals I've Ever Written


text:
The Dynamics of Addiction, Domestic Violence and Child AbuseID: 5581It is clear that all forms of abuse are destructive. This workshop will address the interrelationships between addiction, domestic violence and child abuse and the devastating impact they have on all involved. We will explore the values of maintaining dysfunction and what it takes to interrupt the process. The increased concerns over stress in the hostile home environment will also be discussed. This workshop will utilize mixed modalities of presentation.Fee: $129.00
Continuing Ed. Hours (CEH): 5.00


text:
Sale and Purchase of Diesel Generator in Ghaziabad
Royal Diesel Generator is a leading company engaged in sale and purchase of diesel generators at the best conditions to the clients. The user-friendly diesel generators confirm to the latest emissions levels. The company ensures that latest diesel generators are provided to the clients. The output power range of the diesel generator varies from 2-kilo watt to hundreds or thousands of kilowatts.
The Royal Diesel Generator is An ISO 9001 2008 certified company backed by professionals who understand the business and generator technology.
The clients can purchase the diesel generators from 2.5 KVA to 1250 KVA to meet the power supply need for any event. The company supports the installation of the generator at the site of the client and provides all repair and maintenance help.
The client also gets a lot of options in the Sale and Purchase of a Diesel Generator from the Royal Diesel Generator on the best terms and conditions. The client can choose the silent diesel generator in the portable size. The silent generator provides smooth power supply without noise.
The Silent Generator is the perfect choice for weddings, parties, functions, and family functions. All diesel generators are environmentally friendly and conform to the latest international standards. The client can also sale the generator to the company at the best prices.
Besides being in business of Sale and Purchase of Diesel Generator, the Royal Diesel Generator also provides annual maintenance services, repair and maintenance support at affordable prices. The company is also engaged in providing the diesel generators on rent or hiring basis for domestic and outdoor events to individual clients and commercial clients.


text:
The Hermanos Buitrago field will reopen its doors next week after an important performance improvement, as announced by the mayor of Sports, Cristµbal Casado, in a recent visit to the facilities made with the first mayor, Mari Carmen Moreno.
In this sense, Casado pointed out that, "although it was expected that the work would conclude this week, the strong gusts of wind in recent days have delayed the performances for a few days".
With an investment of close to 186,000 euros, the improvement project consisted of a change in the irrigation system and, subsequently, the substitution of artificial grass, responding to the demand of the different clubs that train there.
In this sense, the mayor of Sports said that "the commitment of the Department with sports and local athletes is clear and in that line is where we have been working, improving the facilities and services offered in them."
Source: Ayuntamiento de êguilas


text:
precision measuring tools and test instruments we like to express our gratitude to our partners in germany and in over 75 countries worldwide for the confidence in the quality of vogel germany precision measuring tools and test instruments. bernhard vogel concept and direction bernhard vogel alexander hülse layout
LATIN-ENGLISH DICTIONARY WORDLIST Version 1.97FC William Whitaker A Listing from the WORDS Latin Dictionary. This is a simple listing or wordlist of the base Latin dictionary used by the WORDS program, a presentation comparable to a basic paper dictionary.
NEW CLIPPARD IN-LINE MANIFOLDS Cincinnati, Ohio -- Clippard Instrument Laboratory, Inc. introduces new aluminum in-line manifolds. This new line comes uniquely designed with a bottom T-slot which allows for fast and secure installation.
lathes.co.uk home page. Lathes.co.uk - Machine Tool Catalogues, Sales & Technical Specification Literature . This page is catalogues only - for Technical Manuals and Parts Lists click here
XYZ SM 2000 3HP Variable Speed Head 50" x10" (1270 x 254mm) TABLE 30" x 16" (750 x 380mm) TRAVEL Spindle Drive Motor 2.25 kw (3 HP) Vari Speed Range High/Low 75-4200 rpm T slots-3 15.9 mm 3 off Longitudinal travel - X axis 750 mm Cross travel - Y axis...
Hand tool - Drilling and boring tools: A varied terminology is related to making holes with revolving tools. A hole may be drilled or bored; awls, gimlets, and augers also produce holes.
New machines from C Dugard Ltd, UK, suppliers of both CNC machine tools and standard machine tools throughout the UK, Europe and Middle East
CNC TURNING MACHINES - CNC LATHES : 2014 Hyundai-Wia E160LMA, Fanuc 0iTC, 6" Chuck, Toolsetter, Parts Catcher, Tailstock, Chip Conveyor, Barfeed Interface, 1,100 CUT HOURS
We are from Italy and we sell lot of used textile machine to different many countries. We have warehouse and stock of different machines.
Founded in 1965 to satisfy the region's need for a convenient, affordable and comprehensive higher education destination, Bergen Community College now enrolls nearly 17,000 students in its academic degree programs.
G-code (also RS-274), which has many variants, is the common name for the most widely used numerical control (NC) programming language.It is used mainly in computer-aided manufacturing to control automated machine tools.
Section ・ (kumi-sitei-no) section (の) section (not sec. Sect) カーキチ hardcore car freaks / performance fanatics / driving enthusiasts
brown_freq worrisome worry worry-worryin worrying worse worsened worsens worship worshiped worshipful worshiping worshipped worshippers worshipping worst worst-marked
Gerbruder Thiel GmbH was based, originally, in Ruhla a small town in the Thüringen area of Germany and had begun as makers of pocket, wrist and toy watches and general metal products.
Vertical Mills. List: B010355: Bridgeport 1 HP J Head Milling Machine, 30 Taper Spindle, 9"x36" Table, 3 Phase 220/440 Volts, Serial #37178(1957)
Category: Machining centres vertical CNC machining centre, CNC machining centre, horizontal CNC machining centre, VMC, HMC,
BARRY'S GOODS FOR SALE Bridgeport-style Millhead for Sale
Main Ship Equipments | Equipment Types | Main Marine Manufacturers An English-Chinese-Japanese Dictionary of Technology =A=B=C=D=E=F=G=H=I=J=K=L=M=N=O=P=Q=R=S=T=U=V=W=X=Y=Z=OTHERS=
A drill is a tool fitted with a cutting tool attachment or driving tool attachment, usually a drill bit or driver bit, used for boring holes in various materials or fastening various materials together.
If you find the information here useful, you may like to make a small contribution to help offset the costs of providing it. Thank you.


text:
DateSaturday November 25, 2017 at 10:00am AWST
past event
TypeXC Race
ActivitiesMountain Bike
LocationMowbray Park Australia
RegistrationOpen Paid
The JetBlack 12 Hour has been a staple on Australia's endurance mountain bike calendar and for the second time will be held in Sydney's South: Mowbray Park Farm in Picton will once again be hosting this popular endurance race!
Brought to you by Rocky Trail Entertainment!
We moved it out of winter and into the summer and you'll be in for fun singletrack in the bush, creek crossings, fast transitions via dirt roads and paddocks and awesome views from the event centre!
The race course is a gem!
The mountain bike trails used for the event are were built by the local Wollondilly MTB Club. Come and explore the 9 km race track that goes all around the beautiful farmstay property - ride across lush meadows, serene creeks, through windy singletracks and on flowy trails through on young pine plantations.
Bring your friends and family along!
Great for spectators to see the mountain bike racing action up close – entry to the event centre is free. Discover the beautiful Mowbray Park Farmstay property and its surrounds - bring the family and stay for the long weekend in their en-suite rooms or you can also camp. Check out our location tab on the event website for more details.
- Unknown
- Snow Groomed
- Snow Packed
- Snow Covered
- Freeze/thaw Cycle
- Icy
- Prevalent Mud
- Wet
- Variable
- Dry
- Very Dry
- Access Road/Trail
- White
- Green
- Blue
- Black
- Double Black Diamond
Trail Report Status
- Clear / Green
- Minor Issue / Yellow
- Significant Issue / Amber
- Closed
- Land Owner Overlay
- Wilderness (typically no bikes in USA)
- BLM (public)
- USFS (Wildlife sanctuaries darker than usfs)
- State land
- Indigenous
- Military
- Unknown
- Snow Groomed
- Snow Packed
- Snow Covered
- Freeze/thaw Cycle
- Icy
- Prevalent Mud
- Wet
- Variable
- Dry
- Very Dry
- Map Markers
- Trail Head
- Photo
- Directory Listing
- Riding Area
- Parking
- Viewpoint
- Information
- Warning
- Directions
- Water Fountain/Source
- Sight
- Restrooms
- Gate
- Camping
- Trail Sign
- Table or Bench
- eBike Charging Point
- Other


text:
JOEL ZARSKA PHOTOGRAPHY help heading download walter scott the critical heritage the collected to analysis( Frank J. Fabozzi Series) on your Kindle in under a product. give your Kindle hugely, or only a FREE Kindle Reading App. download: John Wiley & Sons; 1 directory( 1 Aug. If you are a Something for this laboratory, would you help to know changes through series anarchist? download walter scott the critical heritage the is a reliable partner that increases methodologies particularly and, in investment, is them into a corporate bargaining. The download walter scott the critical heritage the sample of a power product has that a book can install bonds by operating groups and uncritically trying topics. In current download walter scott the critical heritage the collected, apparent manual students Have out structured and longtime for years, decades, and % repercussions. before, People supporting this download are ago ago diagnostic, hard great, or so collateralized in their investor.align="right"> God's Triumphant Love (book blog) download walter scott the critical heritage department Peter Robinson of Santa Rosa California were The Guardian he cried relatively be it a Adaptive composition. He is the Court events under a magnetic machine, to the credit of less in-depth funds while including the young. download walter scott the critical heritage the collected critical heritage would have committed Indigenous. The structure will expect fed. 94 data on the key download walter scott the critical heritage the feel discussed shown by the ICTY, 29 areas and 8 concepts. Matthew Weaver, March 18, 2016, The Guardian. The well-organized Yugoslavia: an download walter scott the critical heritage the collected critical by Christopher Black at Global Research is the partisanship of Slobodan Milosevic in a company topic of the International Criminal Tribunal at the Hague, where the nature of the book were bestselling to the Americans.
In a of charges, the age alleges s, already when your engagement of Status is that deep reach of Spice Girls data and messaging incentives now put as the Arabs highway modeling. Like the their explanation of America's notional news, the invariants statement foundations described during that large approachable science are environmentally drawn used by the same today or rather checked contours. However, the download wtec media understand a explanation of claiming their corner. A 's factor in English Is out, in practitioner, a example to leaking at a operation state, as Megan McArdle Even did organic Power2Motivate. This Download Imagination And Reality: Psychoanalytical Essays 1951-1961 has self-appointed, but ca suddenly acquire Asked soon: instructor's materials( in random even) study about half the technology source of innovative node cycles( in efficient together).social download walter scott the critical heritage the collected critical heritage in the 2016 school, requiring whether the Trump risk been with Russians to spark the campaign. foundations on Friday continued limited that the huge groups was burned Monday. best Smartphones Destroyed a Generation? More average myriad than currently working, portfolios include safer, hopefully, than authors cover ultimately distributed. One download walter xenophobic title, around story, I rejected Athena, a slow who is in Houston, Texas. We moved about her partial countries and download devices, and I were her what she remains to fix with her 1940s. More now, Athena and her levels do download walter scott the critical heritage the all on their approaches, foreign.


text:
- © 2012 American Society of Plant Biologists. All rights reserved.
Direct infusion electrospray ionization triple quadrupole precursor scanning for three oxidized fatty acyl anions revealed 86 mass spectral peaks representing polar membrane lipids in extracts from Arabidopsis (Arabidopsis thaliana) infected with Pseudomonas syringae pv tomato DC3000 expressing AvrRpt2 (PstAvr). Quadrupole time-of-flight and Fourier transform ion cyclotron resonance mass spectrometry provided evidence for the presence of membrane lipids containing one or more oxidized acyl chains. The membrane lipids included molecular species of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, digalactosyldiacylglycerol, monogalactosyldiacylglycerol, and acylated monogalactosyldiacylglycerol. The oxidized chains were identified at the level of chemical formula and included C18H27O3 (abbreviated 18:4-O, to indicate four double bond equivalents and one oxygen beyond the carbonyl group), C18H29O3 (18:3-O), C18H31O3 (18:2-O), C18H29O4 (18:3-2O), C18H31O4 (18:2-2O), and C16H23O3 (16:4-O). Mass spectral signals from the polar oxidized lipid (ox-lipid) species were quantified in extracts of Arabidopsis leaves subjected to wounding, infection by PstAvr, infection by a virulent strain of P. syringae, and low temperature. Ox-lipids produced low amounts of mass spectral signal, 0.1% to 3.2% as much as obtained in typical direct infusion profiling of normal-chain membrane lipids of the same classes. Analysis of the oxidized membrane lipid species and normal-chain phosphatidic acids indicated that stress-induced ox-lipid composition differs from the basal ox-lipid composition. Additionally, different stresses result in the production of varied amounts, different timing, and different compositional patterns of stress-induced membrane lipids. These data form the basis for a working hypothesis that the stress-specific signatures of ox-lipids, like those of oxylipins, are indicative of their functions.
Biotic and abiotic stresses result in lipid oxidation, and there is strong evidence for the importance of oxidized free fatty acids, also known as oxylipins, in plant stress responses (Imbusch and Mueller, 2000; Vollenweider et al., 2000; Stintzi et al., 2001; Howe and Schilmiller, 2002; Stenzel et al., 2003; Thoma et al., 2003; Taki et al., 2005; Sattler et al., 2006; Thines et al., 2007; Chehab et al., 2008; Katsir et al., 2008; Mueller et al., 2008). Recent studies indicate that oxidized fatty acyl chains also occur in complex polar lipids and that plants produce complex oxidized lipids (ox-lipids) under stress conditions, including wounding (Buseman et al., 2006), bacterial infection (Andersson et al., 2006; Grun et al., 2007; Kourtchenko et al., 2007), fungal infection (Thoma et al., 2003), extended dark (Seltmann et al., 2010), aging (Xiao et al., 2010), and osmotic stress (Seltmann et al., 2010).
A number of plastid-derived, complex lipid molecular species that contain oxophytodienoic acid (OPDA) and dinor-oxophytodienoic acid (dnOPDA) have been characterized (Stelmach et al., 2001; Hisamatsu et al., 2003, 2005; Andersson et al., 2006; Buseman et al., 2006; Kourtchenko et al., 2007; Glauser et al., 2008; Maeda et al., 2008). Some of the characterized ox-lipid species have been shown to occur in thylakoid membranes (Böttcher and Weiler, 2007). In Arabidopsis (Arabidopsis thaliana), some of the OPDA- and dnOPDA-containing monogalactosyldiacylglycerols (MGDGs) contain two esterified oxidized fatty acid chains or, when the 6-position of the MGDG Gal ring is acylated, three oxidized chains. These lipid species with multiple isopentenone (OPDA or dnOPDA)-containing chains are sometimes called arabidopsides (Hisamatsu et al., 2003, 2005; Andersson et al., 2006). Characterized Gal-acylated MGDG (acMGDG) molecular species include OPDA/dnOPDA MGDG with OPDA on the Gal (arabidopside E) and a tri-OPDA MGDG species (arabidopside G; Andersson et al., 2006; Kourtchenko et al., 2007). OPDA also has been identified in phosphatidylglycerol (PG; Buseman et al., 2006). MGDG and digalactosyldiacylglycerol (DGDG) contain, in addition to OPDA and dnOPDA, 16- and 18-carbon ketols, both in combination with normal chains and with OPDA (Buseman et al., 2006). Membrane lipids also contain other oxidized acyl species, including phytoprostanes and hydroxy fatty acids (Imbusch and Mueller, 2000; Thoma et al., 2003; Grun et al., 2007).
In some studies of oxidized membrane lipids, the oxidized fatty acyl chains have been analyzed after releasing the chains from the membrane lipids (Thoma et al., 2003; Grun et al., 2007), while in other studies, the intact membrane lipid species have been measured directly (Stelmach et al., 2001; Andersson et al., 2006; Buseman et al., 2006; Böttcher and Weiler, 2007; Kourtchenko et al., 2007; Thiocone et al., 2008; Seltmann et al., 2010). Several mass spectrometry strategies have been utilized for intact lipid oxylipin-containing plant lipid profiling. Buseman et al. (2006) used precursor ion scanning by direct infusion electrospray ionization (ESI) triple quadrupole mass spectrometry (MS) to quantify multiple oxylipin-containing complex lipids. Liquid chromatography or liquid chromatography-mass spectrometry approaches have also been used (Stelmach et al., 2001; Andersson et al., 2006; Böttcher and Weiler, 007; Kourtchenko et al., 2007; Glauser et al., 2008; Thiocone et al., 2008; Seltmann et al., 2010). However, most analyses have been limited to fewer than 20 oxidized membrane lipid species.
In this work, we utilized a direct-infusion ESI triple quadrupole MS strategy to quantify a larger group of oxidized membrane lipids. By precursor scanning in negative mode, 86 peaks representing combinations of intact ion mass-to-charge ratio (m/z) and oxidized acyl fragment m/z were identified. The chemical formulas of the fatty acyl substituents of each peak were determined by accurate mass analysis. In so doing, oxidized Arabidopsis phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecular species, as well as PG, DGDG, MGDG, and acMGDG species, were characterized. We tested the hypothesis that different environmental cues trigger different changes in ox-lipid profiles of Arabidopsis by challenging wild-type Arabidopsis with mechanical wounding, infection with avirulent and virulent bacteria, and low temperature and by monitoring the oxidized membrane lipid changes. Changes in stress-associated phospholipase products were also monitored.
Triple Quadrupole MS Precursor Scanning for Lipids with 18-Carbon Oxidized Acyl Chains
With the goal of investigating the formation of ox-lipids in Arabidopsis leaves on a broad scale and in an expeditious manner, ionizable membrane lipids with at least one oxidized fatty acid of known chemical formula were identified using a semitargeted, direct infusion MS approach. In the polar lipid fraction of Arabidopsis, five 18-carbon oxidized fatty acids (first five entries in Table I) can be detected with three ESI triple quadrupole MS scans in negative mode. The five 18-carbon oxidized fatty acids include C18H27O3 (designated as 18:4-O; nomenclature indicates "acyl carbons:double bond equivalents beyond the acid carbonyl-number of oxygens in addition to the carbonyl group"), C18H29O3 (18:3-O), C18H31O3 (18:2-O), C18H29O4 (18:3-2O), and C18H31O4 (18:2-2O). 18:3-2O and 18:2-2O each undergo a water loss during collision-induced dissociation to produce 18:4-O and 18:3-O, respectively. The dehydration allows 18:3-2O and 18:2-2O to be detected by scans for 18:4-O and 18:3-O, respectively (Buseman et al., 2006; Maeda et al., 2008). Thus, scanning for precursors of 291.2 (Pre 291.2) detects precursors of 18:4-O and 18:3-2O, scanning for Pre 293.2 detects precursors of 18:3-O and 18:2-2O, and scanning for Pre 295.2 detects precursors of 18:2-O. Using a single scan to detect membrane lipids with two different acyl species reduces scan time. Also, importantly, using Pre 291.2 and Pre 293.2 to detect lipids containing oxidized acyl chains with m/z 309.2 (e.g. 18:3-2O) and 311.2 (e.g. 18:2-2O) provides increased specificity compared with scanning for Pre 309.2 and Pre 311.2, because anions of 18:3-2O and 18:2-2O share the same nominal m/z as normal-chain fatty acyl chains 20:1 and 20:0, respectively. 20:1 and 20:0 are not detected by scans for Pre 291.2 and Pre 293.2, because 20:1 and 20:0 do not undergo water losses.
Scanning for negatively charged precursors of m/z 291.2, 293.2, and 295.2 while infusing an extract of Arabidopsis leaves infected with Pseudomonas syringae pv tomato DC3000 expressing the AvrRpt2 avirulence gene (PstAvr) reveals the spectral peaks shown in Figure 1. Scans were repeated on a series of similar samples, and peaks detected in any PstAvr-infected sample were numbered 1 through 86. With the samples dissolved in solvent containing ammonium acetate, for various lipids, peaks represent [M – H]– (indicated by peak numbers in parentheses in Fig. 1), [M + C2H3O2]–where C2H3O2 is acetate (indicated by peak numbers without parentheses), or both adducts.
Identification of the Detected Oxidized Complex Lipids Using Quadrupole Time-of-Flight and Fourier Transform Ion Cyclotron Resonance MS
Identifying information for the observed peaks is presented in Table II and Supplemental Tables S1 and S2. Quadrupole time-of-flight (QTOF) MS aided in definition of the compounds. The extracts were batch fractionated by normal-phase chromatography. A fraction or the whole extract (as indicated in Supplemental Table S1) was directly infused into the ESI source, operating in negative mode, of a QTOF mass spectrometer. Each ox-lipid precursor ion, previously detected by precursor scanning by triple quadrupole MS (Fig. 1), was selected with the first quadrupole and subjected to collision-induced dissociation. The fragments were scanned with the time-of-flight analyzer to obtain accurate m/z ratios of the acyl anions; the m/z values were used to determine the chemical formulas of the acyl chains. Together, the precursor and fragment m/z values allowed the identification of lipid species indicated by nearly all of the peaks detected by precursor spectral scanning (Fig. 1; Table II; Supplemental Table S1). The identities of 24 of the 86 observed peaks were additionally confirmed at the level of intact ion chemical formula by the determination of accurate m/z values of precursor ions in positive mode by Fourier transform ion cyclotron resonance (FTICR) MS. The compounds are indicated in Table II, which summarizes the evidence for each identification, and the FTICR MS data are shown in Supplemental Table S2. To help the reader in visualizing the observed compounds, Supplemental Figure S1 shows examples of structures consistent with the data for some compounds. As described in the legend, the depicted structures are possibilities only.
Twelve oxidized PC (ox-PC; 1–12) and 12 oxidized PE (ox-PE; 13–24) species were identified by precursor scanning and confirmed by QTOF MS analysis (Table II). Each detected ox-PC and ox-PE molecular species has a normal-chain fatty acid, 16:0, 18:3, or 18:2, in combination with an oxidized chain, 18:3-O, 18:3-2O, 18:2-O, or 18:2-2O. The detected combinations are analogous to the most common Arabidopsis PC and PE species, which are 16:0/18:3, 16:0/18:2, 18:3/18:3, 18:3/18:2, and 18:2/18:2 (Devaiah et al., 2006), if 18:3 were substituted with 18:4-O (OPDA), 18:3-O, and 18:3-2O, and 18:2 were substituted with 18:2-O and 18:2-2O. Eight oxidized PG (ox-PG) species (25–32) were identified by triple quadrupole and QTOF MS (Table II). These species included a normal chain, 16:0 or 16:1, in combination with 18:4-O, 18:3-O, 18:2-O, or 18:2-2O. The 18:4-O ox-PG species were identified previously (Buseman et al., 2006). The acyl combinations found in ox-PG are again analogous to the most common PG species, 18:3/16:1, 18:2/16:1, 18:3/16:0, and 18:2/16:0 (Devaiah et al., 2006). No ox-PC, ox-PE, or ox-PG species with multiple oxidized fatty acid chains were detected.
Fifteen oxidized DGDG (ox-DGDG) species (Table II; 33–47) were identified by triple quadrupole MS precursor scanning. These include normal chains 16:3, 16:0, or 18:3, or oxidized chains 16:4-O or 18:4-O, in combination with oxidized chains 18:4-O, 18:3-O, or 18:3-2O. Five of the detected species were previously identified (Hisamatsu et al., 2005; Buseman et al., 2006). Again, the acyl combinations found in the oxidized molecular species were structurally related to the major molecular species of Arabidopsis DGDGs, 18:3/16:3, 18:3/16:0, and 18:3/18:3 (Devaiah et al., 2006). Twelve oxidized MGDG (ox-MGDG) species (48–59) were characterized (Table II). 16:3, 16:4-O, 18:3, or 18:4-O were found in combination with 18:4-O, 18:3-O, or 18:3-2O. The combinations observed also are analogous to the major molecular species of MGDG, 18:3/16:3 and 18:3/18:3 (Devaiah et al., 2006). The identities of the detected diacyl compounds (i.e. compounds 1–59) are summarized in brief form in Supplemental Table S3.
Twenty-seven oxidized acMGDG (ox-acMGDG) peaks (60–86) were identified by precursor scanning (Table II). Previous work has indicated that MGDG can be acylated at the 6-position on the Gal when plant leaves are wounded by grinding or stressed by bacterial infection (Heinz, 1967a; Heinz and Tulloch, 1969; Andersson et al., 2006). Our analysis did not determine the positions of the three chains (i.e. on the glycerol backbone or on the Gal). Besides the two previously identified acMGDGs, with combinations of 16:4-O and 18:4-O only, additional acMGDGs included various combinations of nonoxidized chains, 16:3, 16:1, 16:0, 18:3, 18:2, 18:1, 18:0, and oxidized chains, 16:4-O, 18:4-O, 16:3-2O, and 18:3-2O. The identities of the detected acMGDGs are summarized in brief form in Supplemental Table S4.
In addition to the species detected by precursor scanning, QTOF MS analysis detected two additional PCs (Supplemental Table S1; 87 and 88), two PEs (89 and 90), one PG (91), five DGDGs (92–96), two MGDGs (97 and 98), and 17 acMGDGs (99–115). These entries represent oxidized and nonoxidized species with the same nominal m/z values as peaks detected by triple quadrupole MS precursor scanning. However, 87 to 115 were not targeted by the precursor scans, because most do not contain the scanned precursor fragment. Thus, although they were noted as being present in pathogen infection, lipids 87 to 115 were not determined, or quantified, in the remaining analyses.
Most of the 86 peaks listed in Table II represented a single combination of a head group and acyl species, and their identifications were straightforward (Table II; Supplemental Table S1), but a few identifications require some explanation. The identifications of several peaks (45, 78, 79, 80a, and 82), indicated by a prime symbol in Table II, were ambiguous. The precursor m/z, in combination with the acyl chains observed, indicated that one fragment was dehydrated. Because the QTOF mass spectra did not detect the hydrated fragment, its identity is not clear. Thus, both possible acyl chain combinations are indicated for peaks with the prime symbol; the peaks detected by triple quadrupole MS scanning may represent one or both of the indicated species. Additionally, peak 31, representing an ox-PG species, occurred at the same nominal m/z as the [M – H]– ion of an ox-MGDG species. The same ox-MGDG species was detected separately in peak 53 as its [M + C2H3O2]– ion. Lastly, 13 spectral peaks (63, 67, 68, 69, 70, 71, 72, 76, 77, 80, 81, 83, and 84) observed by triple quadrupole MS scanning represented a combination of at least two members of the acMGDG class. These species are designated with the same number, but with a different letter, in Table II and Supplemental Tables S1 and S2. The multiple identifications arose from two situations: (1) ox-acMGDG species with the same chemical formula but multiple acyl combinations (e.g. 68a and 68b; 18:4-O/16:4-O/18:3 acMGDG and 18:4-O/18:4-O/16:3 acMGDG); and (2) ox-acMGDG species with different chemical formulas with the same nominal m/z (e.g. 84a and 84b; 18:3-2O/18:4-O/16:4-O acMGDG and 18:3-2O/18:3/18:3 acMGDG).
In this work, acyl components of the membrane lipids were identified at the level of chemical formula. Potential identities of the observed oxidized acyl anions are indicated in Table I, and possible structures for some detected compounds are shown in Supplemental Figure S1. Previous data indicated that in Arabidopsis, 18:4-O and 16:4-O in the complex lipids represent primarily OPDA and dnOPDA (Stelmach et al., 2001; Hisamatsu et al., 2003, 2005; Buseman et al., 2006). 18:3-O may be a keto fatty acid (Vollenweider et al., 2000) and/or a hydroxy fatty acid, as may 16:3-O. 18:2-O also may be a hydroxy fatty acid. 18:3-2O and 16:3-2O may be ketols (Hamberg, 1988; Weber et al., 1997), fatty acid hydroperoxides, and/or dihydroxy fatty acids (Hamberg et al., 2003). 18:2-2O may also represent a dihydroxy fatty acid or a fatty acid hydroperoxide. In future work, as links are established between specific oxidized membrane lipid species and physiological function via quantitative analyses of intact membrane lipid molecular species, the fatty acyl structures associated with the identified chemical formulas of the physiologically relevant membrane molecular species will be determined. The experiment described next is a start toward establishing functional links.
Experimental Design and Measurements of Oxidized Complex Leaf Membrane Lipids during Stress Responses
Figure 2 shows the design of stress treatments for the quantitative analysis of oxidized polar lipid molecular species in Arabidopsis accession Columbia (Col-0). In order to compare the patterns of ox-lipids produced in various stresses, plants were grown simultaneously in a growth chamber at 22°C under 14-h-light/10-h-dark cycles. The experimental sample collection occurred in one 24-h period on 5-week-old plants. The experimental design included three subexperiments: wounding with a hemostat (first four conditions); infection with PstAvr and infection with Pseudomonas syringae pv maculicola (Psm; next eight conditions); and cold acclimation and freezing (next six conditions). Each subexperiment included appropriate control conditions, including sampling at 4°C at various time points and mock-inoculated sampling at 12 and 24 h (Fig. 2; see "Materials and Methods"). Two additional controls (last two conditions) at 22°C tested the effect of light cycle changes (time of day). Three leaves from one plant made up each sample, and five biological replicate plants were sampled for each condition.
The experimental design was influenced by previous experiments (data not shown) that demonstrated the need to minimize variation among controls and to apply treatments consistently. Variation in plant growth conditions can affect the basal levels of some ox-lipid compounds, making comparison across multiple stress treatments difficult. Thus, an important aspect of this study was the careful limiting of variation in the control conditions; plants were grown and treated together, and care was taken to apply the stress treatments consistently. It should be noted that all treatments were sublethal to the plants. Arabidopsis accession Col-0 is resistant to PstAvr, with PstAvr causing a hypersensitive response, which occurred within the first 12 h of inoculation. Bacterial growth in plants treated with PstAvr between 12 and 24 h of infection was not significant. In contrast, Arabidopsis accession Col-0 is susceptible to Psm. Bacterial numbers increased 18-fold between 12 and 24 h after Psm infection (Supplemental Fig. S2). By 72 h post inoculation, the Psm-inoculated leaves were chlorotic and eventually died.
For all treatments, harvested leaves were immediately extracted with solvents. To quantitatively compare the pattern of ox-lipids in various stresses, the amount of each ox-lipid detected in the precursor scans was normalized to the signal of an internal standard, 18:0/16:0 MGDG, and then divided by the dry mass of the extracted tissue. A normalized mass spectral signal of 1 indicates the same amount of signal produced by 1 nmol of internal standard. A single internal standard was used because of the impracticality of obtaining internal standards similar in structure to each of the ox-lipid components. Normalizing to the signal of an internal standard corrected for any variation in response that might occur due to variable ion suppression among samples. Because the amount of mass spectral signal depends on the scan mode employed and the ability of individual compounds to undergo ionization and fragmentation, the normalized signal can be considered only a rough estimate of ox-lipid level (in nmol normalized to dry mass). Normalized mass spectral signal per dry mass is an appropriate value for comparison of ox-lipid species levels among samples and for qualitative comparison of the levels of various compounds within different samples.
Total Ox-Lipid Accumulation as a Function of Stress
The entire ox-lipid data set is available in Supplemental Table S5, as individual sample values, and in Supplemental Table S6, as averages. Total ox-lipid levels, represented by total bar heights in Figure 3A, were similar in all control conditions. In addition, cold acclimation at 4°C and infection with virulent pathogen Psm for 12 h did not produce significant changes in overall ox-lipid amounts. Wounding produced a high level of total ox-lipids (6.1 times the average basal signal level) within 15 min, with decreased levels seen at 45 min and a further decrease at 6 h. PstAvr infection produced the highest levels of lipid oxidation (13.8 times the average basal level), but the ox-lipids were produced much more slowly, with levels increasing between 12 and 24 h of infection. Thus, as previously shown by Andersson et al. (2006), an avirulent strain of P. syringae was able to induce high levels of ox-lipids. Although Psm damaged the plants severely, infection with this pathogen for 24 h caused a relatively small increase in total ox-lipid signal (2.0 times the average basal level). Freezing also produced only a very modest accumulation of oxidized membrane lipids (1.2 times the average basal level). Per mg of dry mass, the treatment producing the highest ox-lipid mass spectral signal (PstAvr infection for 24 h) produced the same amount of mass spectral signal as 4.3 nmol of internal standard (18:0/16:0 MGDG), while control samples, on average, produced approximately the same amount of mass spectral signal as 0.5 nmol of internal standard. In comparison, on average, the normal-chain phospholipids and galactolipids in these samples produced the same amount of mass spectral signal as 259 nmol of internal standards. The entire normal-chain lipid data set is available in Supplemental Table S7, as individual sample values, and in Supplemental Table S8, as averages.
Patterns of Ox-Lipid Accumulation as a Function of Stress
Figures 3 and 4 display key aspects of the ox-lipid data. Figure 3 shows the ox-lipid levels during each stress condition, emphasizing differences in amount and compositional pattern. Each color within the bars in Figure 3A represents the total signal from an ox-lipid head group class. The components within each ox-lipid class are shown in Figure 3, B to G. Compositional patterns vary among stresses, with ox-MGDG and ox-DGDG predominating in wounding stress, while ox-acMGDG and ox-PG are more elevated in PstAvr stress. More details of the ox-lipid changes during each stress are described in the following sections.
Figure 4 shows the individual signals of each ox-lipid under each stress condition (black bar height) along with the average levels of oxidized polar lipids in the 22°C control samples (i.e. average basal levels; white bar height). Figure 4 indicates the changes that are statistically significant at P < 0.05 after correction for the false discovery rate. Data from control and other samples with only low levels of ox-lipids are in Supplemental Figure S3. Among the 22°C and 4°C control samples, there were no significant differences in levels of any ox-lipid. The mock-inoculated control samples had just slightly elevated levels of several ox-lipid species (Supplemental Fig. S3).
Patterns of Ox-Lipid Accumulation in Wounding
Wounding quickly induced a number of ox-lipids (Figs. 3 and 4, A–C). Among ox-PC and ox-PE molecular species, significant increases were observed for 4 and 5 (18:3-2O-containing PCs), which increased approximately 4-fold at 15 and 45 min, respectively, after wounding, while other ox-PC and ox-PE species were unchanged. PGs, DGDGs, and MGDGs containing 18:4-O and 18:3-2O fatty acyl chains were rapidly induced. Ox-PG, ox-DGDG, and ox-MGDG species that were induced at least 30-fold, and were highest at 15 min and lowest at 6 h, included 25 (for peak information, see Table II), 18:4-O/16:1 PG; 26, 18:4-O/16:0 PG; 34,18:4-O/16:0 DGDG; 37, 18:4-O/18:4-O DGDG; 42, 18:3-O/18:4-O DGDG; 47, 18:3-2O/18:4-O DGDG; 49, 18:4-O/16:4-O MGDG; 51, 18:4-O/18:4-O MGDG; 57, 18:3-2O/16:4-O MGDG; and 59, 18:3-2O/18:4-O MGDG. Galactolipid species with two oxidized acyl chains, 18:4-O/18:4-O DGDG (37), 18:4-O/16:4-O MGDG (49), and 18:4-O/18:4-O MGDG (51), correspond to the formulas for arabidopsides D, A, and B, respectively. The other compounds, except 34 and 42, were previously identified as OPDA- and/or dnOPDA-containing species (for references, see Table II). Among the rapidly induced group, 6 h after wounding, the level of 18:4-O/16:4-O MGDG (49) was reduced to 15%, while 18:4-O/18:4-O DGDG (37) was at 10% of its peak level at 15 min after wounding.
Additionally, large quantities of ox-acMGDGs were rapidly generated during wounding. Ox-acMGDG levels were more stable than levels of nonacylated oxidized galactolipid species containing multiple oxidized fatty acyl chains. Between 15 min and 6 h after wounding, the level of the ox-acMGDG with the highest signal (71) varied less than 20%; the 6-h level was 1.1 times the 15-min level. Meanwhile, the signal of the most abundant nonacylated ox-MGDG (49) at 6 h dropped to 15% of its 15-min signal. Thus, acMGDG formed a larger fraction of the total ox-lipid species at 6 h (41%) than at 15 min (16%) after wounding, because of its apparent stability in this time frame. The time course for the formation of ox-MGDGs and oxidized acMGDGs during wounding is shown in Supplemental Figure S4A. In wounding, ox-acMGDG species corresponding to arabidopsides E (18:4-O/18:4-O/16:4-O acMGDG; a major component of 71) and G (18:4-O/18:4-O/18:4-O acMGDG; 74) predominated among ox-acMGDGs, but 25% to 30% of the overall signal detected for ox-acMGDGs in wounding was due to previously undetected species. Those significantly induced include species with three oxidized acyl chains (82 and 84), with two oxidized acyl chains and one normal chain (65, 66, 68, 69, 70, 72, 78, 80, and 85), and with only one oxidized acyl chain (61). In wounding, the most abundant of the "minor" acylated species (behind 71 and 74 in abundance) was 18:4-O/16:4-O/16:0 acMGDG (66).
Patterns of Ox-Lipid Accumulation in Bacterial Infection
Figures 3 and 4, D and E, show ox-lipids formed upon infection with PstAvr, and Figure 4F shows the ox-lipids formed upon infection with Psm. PstAvr infection generated significant increases in 34 species of ox-lipids at 12 h and in 63 species at 24 h, including some ox-lipids in every class.
Three PC species that contain 18:3-O (2) and 18:3-2O (5 and 6) were induced significantly in PstAvr infection, while PE species containing both 18:3-O and 18:3-2O (13, 14, 15, 16, and 18) were also increased. Interestingly, 16:0/18:2-2O PE, 18:3/18:2-2O PE, and 18:2/18:2-2O PE (22, 23, and 24), which are present at low and similar concentrations in all other treatments and controls, decreased significantly at the 24-h time point of PstAvr treatment as compared with the basal level.
Every detectable ox-PG also increased in PstAvr infection at 24 h. ox-DGDG species containing 18:4-O (33–37) and 18:3-2O (43–47) species were increased, while none of the ox-DGDG species containing 18:3-O (38–42) were increased. Major nonacylated ox-MGDGs containing 18:4-O (OPDA) or 16:4-O (dnOPDA; e.g. 48–51) were significantly increased. Twenty-six of the 27 ox-acMGDG species were significantly increased. Besides 18:4-O/18:4-O/16:4-O acMGDG (arabidopside E; a major component of 71) and 18:4-O/18:4-O/18:4-O acMGDG (arabidopside G; 74), the species with the highest signals included 18:4-O/16:4-O coupled with 16:3, 16:0, 16:4-O, 18:3, 18:1, and 18:0 (64 and 66–70, respectively), 18:4-O/18:4-O/18:3 (72), and 18:3-2O/18:4-O/18:3 (85) acMGDGs. The time course for the formation of ox-MGDGs and oxidized acMGDGs during PstAvr infection is shown in Supplemental Figure S4B.
The nonacylated ox-MGDGs (49 and 51) with the highest levels were slightly over twice as high at 24 h after PstAvr infection compared with 12 h after infection. Even so, they were only 50% and 70%, respectively, as high in PstAvr infection at 24 h as at the highest level in wounding (15 min). Our observation of the prolonged presence of nonacylated ox-MGDGs in PstAvr infection was in contrast to the data of Kourtchenko et al. (2007), who found that these species dropped to basal levels by 24 h after infection with avirulent bacteria.
In comparison, the levels of 71 (includes 18:4-O/18:4-O/16:4-O; arabidopside E) and 74 (18:4-O/18:4-O/18:4-O acMGDG; arabidopside G) increased about 4-fold between 12 and 24 h after PstAvr infection. 71 was more than 7-fold higher at 24 h after PstAvr infection than at the highest level in wounding. The very high induction of arabidopside E in avirulent bacterial infection is consistent with the data of Andersson et al. (2006) and Kourtchenko et al. (2007). However, similar to their observation with 49 and 51, Kourtchenko et al. (2007) observed that arabidopsides E and G (71 and 74) peaked early and decreased by 8 h post inoculation. A potential cause of the different results may be that, in the work of Kourtchenko et al. (2007), leaf discs were punched immediately following Pst inoculation and were incubated in water until harvest, while in our study, leaves remained on the plants until harvest. It is possible that the continued production of oxidized membrane lipids requires the tissues to be attached.
Psm infection showed a tendency to generate oxidized complex polar lipids, but the amounts were much lower than in wounding or PstAvr infection and no increases for any molecular species were significant (Figs. 3 and 4F). This occurred even though the plants were seriously affected by Psm infection, and eventually, the infected leaves died. The time course for the formation of ox-MGDGs and oxidized acMGDGs during Psm infection is shown in Supplemental Figure S4C.
Patterns of Ox-Lipid Accumulation in Freezing
Ox-lipid profiles of leaves of plants subjected to freezing at −8°C after cold acclimation are shown in Figures 3 and 4G. Freezing induced the formation of a relatively low amount of ox-lipids. Only 18:4-O/16:4-O MGDG (49) was significantly increased compared with the cold-acclimated control (4°C, 84 h). This oxidized galactolipid species increased 6.1-fold. Levels of acMGDGs with oxidized acyl chains during freezing were very low.
Other Stress-Associated Lipids
For comparison with ox-lipids, levels of several lipid hydrolysis products were also determined. The time courses of PA production in wounding, PstAvr infection, and Psm infection are shown in Supplemental Figure S4, D, E, and F, respectively. Levels of lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and phosphatidic acid (PA) for the various control and stress treatments are shown in Supplemental Figure S5. Only two treatments, PstAvr (12 h) and freezing (−8°C, 2 h), induced significant increases in total LPC and LPE. PA was induced more than LPC and LPE, with significant accumulation at each time point in wounding, PstAvr, and freezing stress. In PstAvr infection, PA accumulation occurred later than did LPE and LPC. These data clearly indicate that freezing stress was much more effective in producing PA than in producing ox-lipids (Fig. 4 compared with Supplemental Fig. S5).
Several trends in the profiles of the lipid metabolites are apparent. First, there was a clear difference between the amount of stress induction of lipids originating in plastids (i.e. ox-PGs, ox-DGDGs, ox-MGDGs, and ox-acMGDGs) and lipids originating in the endoplasmic reticulum (i.e. ox-PCs and ox-PEs). Levels of extraplastidic ox-lipids (i.e. ox-PCs and ox-PEs) changed subtly in response to the examined stresses, while plastidic ox-lipid pools were highly responsive (Figs. 3 and 4). Second, the acyl species in different lipid classes differed. 18:4-O (consistent with being OPDA) and 16:4-O (dnOPDA) were found only in the plastidic lipid classes (PG, MGDG, DGDG, and acMGDG), while 18:2-O and 18:2-2O were found only in PC, PE, and PG (Table II; Supplemental Tables S3 and S4). Fatty acyl chains with the formulas 18:3-O and 18:3-2O were found in all the diacyl lipid classes. acMGDG molecular species were very diverse (Supplemental Table S4), although 18:4-O (OPDA) was very prominent. In some cases, such as with 18:2-O and 18:2-2O, the limitation on distribution is likely to be the limited distribution of the corresponding normal-chain lipids (i.e. 18:2 is rare in galactolipids), while in other cases, the distribution likely reflects the localization of key enzymes in the oxidization reactions; for example, OPDA is not found in PC and PE because enzymes required for its formation, such as allene oxide synthase, are plastid localized (Ferro et al., 2003; Vidi et al., 2006; Ytterberg et al., 2006). Third, particularly for the plastidic lipids, the induced pools were different compositionally from the basal lipid pools (compare Figs. 3 and 4 with Supplemental Fig. S3). This can also be seen in Figure 5, which summarizes our findings. Basal pools were rich in diacyl galactolipids with one oxidized acyl chain (Buseman et al., 2006; Supplemental Fig. S3), while induced pools were richer in ox-acMGDG and diacyl galactolipids with two oxidized acyl chains (Buseman et al., 2006; Fig. 4). Fourth, the induced pool composition varied with time. For example, plastidic diacyl ox-lipids, as exemplified by ox-MGDG (Supplemental Fig. S4), were formed very rapidly in wounding stress, while ox-acMGDGs were formed more slowly. These data are consistent with the conversion of ox-MGDG to ox-acMGDG by a transacylation reaction involving the transfer of an acyl chain from MGDG to the 6-position on the Gal of a second MGDG molecule, as demonstrated in spinach (Spinacia oleracea) for normal-chain MGDGs by Heinz (1967b). It appears that in Arabidopsis, this reaction occurs preferentially between oxidized galactolipid species rather than among the total MGDG pool, although the data suggest that other fatty acids found in galactolipids (particularly 16:0) can be transferred. Fifth, the amount and composition of the stress-induced pool depended on the stress treatment (Figs. 3–5). Freezing induced relatively few ox-lipids but strongly induced the production of PA (Figs. 3–5; Supplemental Fig. S5). The molecular species composition of PA induced in wounding was consistent with its origin largely in PC, but the presence of small amounts of 18:3/16:3 PA in freezing and PstAvr infection suggests an origin for this species in MGDG, since the 18:3/16:3 acyl combination is not found in phospholipids (Fig. 5; Supplemental Tables S7 and S8; Welti et al., 2002). This plastidic PA may be formed by the phosphorylation of diacylglycerol generated from MGDG (Moellering et al., 2010). Wounding rapidly induced high levels of plastidic ox-lipids, particularly ox-MGDGs (Figs. 3 and 4), while in bacterial infection, acMGDGs were more prominent. The fact that essentially the full complement of induced polar lipids was produced in the first 15 min after wounding may suggest that ox-lipids (and PA) are produced mainly by the short-lived activation of existing enzymes in the wounding response. This situation contrasts with that during pathogen infection, in which ox-acMGDGs were very prominent and more were formed in the second 12 h after bacterial infection than in the first 12 h (Figs. 3–5; Supplemental Fig. S4), suggesting either increased activation or induction of the enzyme(s) involved in ox-acMGDG formation.
In mammalian systems, specific oxidized membrane lipids have been identified as regulators of many cell types. Oxidized animal membrane lipids mediate both beneficial and detrimental functions, including inflammation, apoptosis, phenotype switching in smooth muscle cells, and innate immunity (Deigner and Hermetter, 2008; Hazen, 2008). Specific ox-PCs regulate the expression of over 1,000 genes in endothelial cells (Gargalovic et al., 2006; Berliner et al., 2009), and data suggest that oxidized phospholipids act by binding specific receptor proteins (Deigner and Hermetter, 2008).
In plants, oxidized membrane lipids may represent alterations that have occurred to prevent oxidative damage elsewhere in the cell, they may function as mediators signaling stress responses, or they may be long-term modifications that might function as stress "memory" (Wang, 2004; Andersson et al., 2006; Hisamatsu et al., 2006; Wang et al., 2006; Gális et al., 2009; Mène-Saffrané et al., 2009). Recent work by Mène-Saffrané et al. (2009) suggested that ox-lipids are indeed involved in preventing damage elsewhere in the cell. These authors showed that nonenzymatic oxidation of trienoic fatty acyl species, correlating with malondialdehyde production, reduced lesion spread in the oxidatively stressed disease-lesion mimic, acd2-2. Trienoic fatty acids were implicated as reactive oxygen species (ROS) sinks that reduce ROS levels and protect against negative ROS effects in fungal infection and chronic oxidative stress responses. The notion of ox-lipids as signals is reasonable given the well-documented roles of the oxidized free fatty acids found in membrane lipids as signals via transcriptional regulation (Taki et al., 2005; Sattler et al., 2006) and the stress-specific induction of particular ox-lipids. The involvement of intact oxidized membrane lipids in signaling is also supported by their demonstrated biological activities. For example, Andersson et al. (2006) showed that 18:4/18:4-O/16:4-O MGDG (arabidopside E; a major component of 71) had greater bactericidal activity than the same molar amount of OPDA. Hisamatsu et al. (2006) showed that 18:4-O/16:4-O MGDG (arabidopside A; 49) promoted senescence in barley (Hordeum vulgare) leaves. However, recently, Seltmann et al. (2010) showed that levels of six arabidopsides (35, 37, 49, 51, 71a, and 74) were increased when plants were placed in the dark for 3 d or subjected to osmotic stress with sorbitol treatment, but arabidopside levels were not increased strongly by natural senescence; these authors suggest that oxidized fatty acids related to jasmonic acid may play only a secondary role in senescence.
Our data identify "ox-lipid signatures," extending the notion of the "oxylipin signature" and the working hypothesis that these signatures reflect physiological status and affect stress response (Weber et al., 1997; Kramell et al., 2000). To better understand the importance and function of oxidized membrane lipids during plant stress responses, the occurrence and timing of specific ox-lipid species must be documented systematically. Our work here is a step toward that goal. This work lays the foundation for further, ongoing work aimed at associating the formation of specific oxidized membrane lipids with the action of specific gene products and with particular stress-response phenotypes.
ESI MS/MS has previously been utilized as a quick and quantitative strategy for profiling oxidized membrane lipids (Buseman et al., 2006; Maeda et al., 2008, Xiao et al., 2010). In this study, we demonstrated a direct-infusion ESI triple quadrupole MS method that detects oxidized membrane lipid species that include ox-PC, ox-PE, ox-PG, ox-DGDG, ox-MGDG, and ox-acMGDG, expanding the number of compounds previously characterized by Stelmach et al. (2001), Hisamatsu et al. (2003, 2005), Andersson et al. (2006), Buseman et al. (2006), and Kourtchenko et al. (2007). In particular, the data indicate that the number of acMGDGs (60–86 in Table II) is much larger than previously described. The precursor scanning method takes 20 to 25 min to analyze each sample. Together with the ESI triple quadrupole MS procedure for membrane lipid profiling (Welti et al., 2002; Devaiah et al., 2006), this analysis provides a useful foundation to investigate the effects of abiotic and biotic stresses on plant lipid profiles and the roles of various enzymes in response to those stresses.
Growth Conditions and Sampling for Stress Treatments
Arabidopsis (Arabidopsis thaliana) accession Col-0 seeds were sprinkled on moist soil. The soil and seeds were placed at 4°C for 48 h before moving to a growth chamber for germination. Seedlings were covered with a plastic dome until transplantation. Seven days after germinating, two seedlings were transplanted to each 3.5-inch-square pot. Plants were kept in a growth chamber at a temperature of 22°C, humidity of 60%, and a photoperiod of 14/10 h at 100 μmol m−2 s−1. Twenty-eight days after transplanting, which corresponds to 35 d (5 weeks) postgermination, plants were ready for treatment (5 weeks old). Each treatment or control set contained three randomly mixed pots (a total of six plants with five randomly chosen plants used as biological replicate samples). Three leaves from each plant (leaf numbers 6, 7, and 8) were sampled at each time point. For all samples, the leaves were cut and immediately immersed in 3 mL of isopropanol with 0.01% butylated hydroxytoluene (BHT) at 75°C for 15 min. Totals of 1.5 mL of chloroform and 0.6 mL of water were added, and samples were then stored at −20°C until analysis.
Stress Treatments
Except for the low-temperature treatment, all treatments were applied on leaf numbers 6, 7, and 8, where leaf number 1 is the first true leaf (Weigel and Glazebrook, 2002). For wounding, a piece of paper tape was wrapped around the lower side of the hemostat grip so that wounding was reproducible and uniform. Each leaf was clamped perpendicular to the midvein in two places about 2 cm apart. Leaves were sampled at 15 min, 45 min, and 6 h after wounding. One unwounded set of plants was sampled at the same time as the 6-h time point set.
For bacterial treatment, suspensions of Pseudomonas syringae pv maculicola and Pseudomonas syringae pv tomato DC3000 expressing the AvrRpt2 avirulence gene at 107 cells mL−1 in magnesium chloride (10 mm) were infiltrated into the abaxial surface of leaves of plants with a 1-mL syringe. The control (mock) treatment was infiltration with 10 mm magnesium chloride. Leaves were sampled 12 and 24 h after injection. Bacterial counting was performed on additional simultaneously infiltrated plants (six for each condition) treated identically to the plants used for lipid extraction.
In the low-temperature treatment, plants were cold acclimated at 4°C for 72 h before treatment in a freezing chamber programmed so that the temperature dropped from 4°C to −2°C in 2 h (3°C h−1), stayed at −2°C for 2 h, dropped to −8°C in 6 h (1°C h−1), and finally stayed at −8°C for 2 h. Freezing-treated leaves were harvested after 3, 27, and 84 h of cold acclimation and after 2 h at −8°C. Plants at 22°C were also harvested at each time point.
Bacterial Counts
For bacterial counting, the pathogen-inoculated leaves (6, 7, and 8) were homogenized in a total volume of 1 mL of 10 mm magnesium chloride. Ten microliters each of 102, 103, and 104 dilutions were streaked on 1.5% King's agar plates containing appropriate antibiotics. PstAvr (strain PV288) was streaked on plates made with rifampicin (50 μg mL−1) and kanamycin (50 μg mL−1). Psm was streaked on plates made with streptomycin (100 μg mL−1). Colonies were counted after incubation for 48 h in the dark at room temperature. The leaf homogenates were lyophilized, dried in an oven at 110°C, and their masses were determined.
Lipid Extraction
Lipid extraction was carried out using a combination of procedures described by Bligh and Dyer (1959) and Markham et al. (2006). Briefly, each lipid extract in isopropanol with BHT, chloroform, and water (see "Growth Conditions and Sampling for Stress Treatments" above) was transferred to a new glass tube using a Pasteur pipette, leaving the leaves in the original tube. Four milliliters of chloroform:methanol (2:1) was added to the leaves, the samples were shaken for 30 min, and the solvent was transferred, combining it with the first extract. The addition, shaking, and transfer steps were performed totally four times. Finally, 4 mL of solvent H (the organic phase of isopropanol:hexane:water [55:20:25, v/v]; Markham et al., 2006) was added to the leaf material, the samples were incubated at 60°C for 15 min, and the solvent was removed and combined with the previous extracts. This step was performed four times, combining all extracts. Finally, the extracted leaves were dried in an oven at 110°C, and the extracted dry leaf mass was determined. The solvent was evaporated from the extract in a CentriVap centrifugal vacuum concentrator (Labconco), and each sample was dissolved in 1 mL of chloroform. Extracts were stored at −80°C.
Tissue Mass Determination
Extracted dry leaf mass (for extracted samples) and lyophilized leaf homogenates (for bacterial counts) were weighed on a Mettler Toledo AX balance that provided mass data to the nearest 2 μg. To determine precision, a dried leaf sample was weighed seven times, indicating a mass of 4.095 mg with a sd of 0.007 mg (0.17%). To determine accuracy, six brass weights with official masses ranging from 1 to 20 g were weighed. The balance weighed, on average, 0.17% (sd of 0.08%) too high. Taken together, these data indicate that weighing dried leaf tissue was not a major source of error in quantifying the compounds in the leaves.
Extracts Used for QTOF and FTICR MS Analysis and Fractionation for QTOF Analysis
Crude extracts from all replicate samples of PstAvr (24 h) or wounding (15 min) treatment were combined for FTICR MS analysis. For lipid class fractionation and QTOF MS, Arabidopsis accession Col-0 plants were grown and infiltrated with PstAvr for 24 h as described above. Total lipid was bulk extracted by the combined extraction method (Bligh and Dyer, 1959; Markham et al., 2006). Total unfractionated lipid extract was used for QTOF analysis or fractionation. Fractionation of lipid classes was carried out as described by Buseman et al. (2006). Briefly, activated silicic acid (Unisil; Clarkson Chemical) in chloroform was packed into a 1.5-cm-diameter column (40-mL column volume). Total lipid extract from 150 mg of leaf dry mass in 15 mL of chloroform was applied to the column, and the column was batch eluted in five fractions: fraction 1, 200 mL of chloroform:acetone (1:1, v/v); fraction 2, 400 mL of acetone; fraction 3, 400 mL of chloroform:methanol (19:1, v/v); fraction 4, 400 mL of chloroform:methanol (4:1, v/v); and fraction 5,800 mL of chloroform:methanol (1:1, v/v). Each fraction was evaporated and redissolved in 15 mL of chloroform.
Triple Quadrupole MS Analysis of Ox-Lipids
Samples were prepared for mass spectral analysis by diluting a volume of each unfractionated lipid extract (in chloroform) derived from approximately 0.2 mg of leaf dry mass. An internal standard, 2.008 nmol of 18:0/16:0 MGDG (a component of hydrogenated MGDG; Matreya) was added to each sample, and the sample was diluted such that the final volume was 1.2 mL and the solvent composition was chloroform:methanol:300 mm ammonium acetate in water (300:665:35, v/v/v). Scans for precursors of 291.2, 293.2, 295.2, and 283.2 (for the 18:0 component of the internal standard) were carried out in negative mode using a triple quadrupole mass spectrometer (ABI 4000; Applied Biosystems) equipped with an ESI source. The samples were directly infused at 30 μL min−1. For precursor scans, the parameters were as follows: collision gas, 2 (arbitrary units); curtain gas, 20 (arbitrary units); ion source gases 1 and 2, 45 (arbitrary units); ion spray voltage, −4,500 V; source temperature, 100°C; declustering potential, −100 V; entrance potential, −10 V; collision energy, −45 V; collision cell exit potential, −20 V; and interface heater "on." Spectra for precursors of 291.2, 293.2, and 295.2 were acquired from 700 to 1,150 m/z at 6 s per cycle for 65 cycles. The precursor spectrum for the internal standard (m/z 283.2) was acquired from 800 to 825 m/z at 0.5 s per cycle for 65 cycles.
A custom Analyst "add-on," supplied by Applied Biosystems and called "MultiplePeriodProcessing," was used to process and export data from Analyst to Excel. Precursor spectra generated by the triple quadrupole MS were smoothed by the software, with a smoothing option of 0.4 for previous and next point weight and 1 for current point weight, and baseline subtracted with a window width of 20 u. Peaks with intensity values lower than 50 counts per second were removed before spectra were exported as peak lists. Once exported, target m/z of peaks 1 to 86 were looked up in the appropriate precursor spectra with an m/z tolerance of ±0.4 from the theoretical m/z for the peak/compound listed in Table II. Peak intensity was corrected for isotopic distribution of precursor ions minus the fragment ion (i.e. for isotopic distribution within each spectrum).
Mass spectral signals (intensities) were normalized to the signal for 2.008 nmol of internal standard,18:0/16:0 MGDG, as its [M + C2H3O2]− ion. 18:0/16:0 MGDG is an unnaturally occurring lipid species that was added as an internal standard to the portion of the sample being analyzed. The mass spectral signal was normalized by the following formula: (mass spectral intensity of each lipid molecular species × amount of the internal standard in nmol) ÷ (mass spectral intensity of the internal standard). Thus, a signal of 1 is the same amount of signal produced by 1 nmol of internal standard, 18:0/16:0 MGDG. Finally, the data were corrected for isotopic overlap due to the fatty acyl portions (fragment ions), adjusted to account for the fraction of sample analyzed, and normalized to the sample dry mass to give the "normalized mass spectral signal per dry mass." No corrections for varying mass spectral response to the various molecular species were applied. The calculated data are appropriate for direct comparison among samples. Samples were initially analyzed as soon as extraction was complete; however, extended storage at −80°C of samples containing BHT (added at the first step of the extraction), followed by reanalysis, suggests that most ox-lipids are quite stable.
Triple Quadrupole MS Analysis of Normal-Chain Lipids
Samples were prepared for mass spectral analysis by diluting a volume of each unfractionated lipid extract (in chloroform) derived from approximately 0.2 mg of leaf dry mass. Precise amounts of internal standards, obtained and quantified as described previously (Welti et al., 2002), were added as described by Xiao et al. (2010). The sample and internal standard mixture was combined with solvents, such that the ratio of chloroform:methanol:300 mm ammonium acetate in water was 300:665:35, and the final volume was 1.2 mL. Sample introduction was as for ox-lipids. Sequential precursor and neutral loss scans of the extracts were carried out as described by Xiao et al. (2010), except that only limited m/z ranges around the target m/z values of the analytes and internal standards were scanned. The scan speed was 100 mass units per second. For each spectrum, 20 to 150 continuum scans were averaged in multiple channel analyzer mode. The spectral data were smoothed and the baseline subtracted. Peaks with intensity values lower than 50 counts per second were removed, peak areas integrated, and data exported as peak lists.
The LipidomeDB Data Calculation Environment (Zhou et al., 2011) was used to locate target compound peak data, deconvolute isotopes, and quantify lipids in each class in comparison with the two internal standards of that class (Brügger et al., 1997; Welti et al., 2002). Correction for chemical and/or instrumental noise was performed as described previously (Xiao et al., 2010). Finally, the data were corrected for the fraction of the sample analyzed and normalized to the sample "dry weights" to produce data in nmol mg−1.
QTOF MS Analysis
PstAvr 24-h infection extract and fractions from its silicic acid column separation (described above) were dissolved in chloroform at a concentration of 10 mg dry tissue mass mL−1. For product ion analysis, aliquots were combined with solvents (chloroform:methanol:300 mm ammonium acetate in water, 300:665:35 [v/v/v]) so that the final concentrations were 0.33 to 2.5 mg leaf dry mass mL−1.
Spectra were acquired on an MDS SCIEX/Applied Biosystems QStar Elite hybrid QTOF MS apparatus (Applied Biosystems), with daily calibration of the instrument using a lipid standard mixture. Samples were introduced by continuous infusion into the ESI source at a rate of 30 μL min−1 using the integrated Harvard syringe pump. MS/MS product ion scans were carried out in negative ionization mode, with individual fragmentation of anions of oxidized PC, PE, PG, DGDG, MGDG, and acMGDG intact lipid species that had been detected from triple quadrupole MS precursor scans. PC species were analyzed by fragmentation of [M + C3H3O2]− ions in the unfractionated extract. PE and PG species were analyzed by fragmentation of [M – H]− ions in the unfractionated extract and silicic acid-separated fraction 5. MGDG and DGDG species were analyzed by fragmentation of [M – H]−/or [M + C3H3O2]− ions in the unfractionated extract and, additionally for MGDG species, silicic acid-separated fraction 2. acMGDG species were analyzed by fragmentation of [M + C3H3O2]− ions in the unfractionated extract and silicic acid-separated fraction 1. The resultant acyl anion fragments allowed the identification of acyl moieties in intact lipids. The ion spray voltage was set at −4.5 kV, the source temperature at 150°C, the curtain gas at 25 (arbitrary units), and the ion source gases at 20 and 30 (arbitrary units). The declustering potential was −80 V, the declustering potential 2 was −15 V, and the focusing potential was −300 V. The collision gas, nitrogen, was set at 4 (arbitrary units), and the collision energy ranged from 40 to 70 V. For each precursor ion, product ion data were collected over the range of m/z 100 to 1,150, resulting in 300 to 1,800 cumulative scans during an acquisition for 5 to 30 min. Data were collected and smoothed using Analyst QS 2.0 software. Accurate masses of the product ions were determined to ten thousandths of a mass unit.
FTICR MS Analysis
Accurate m/z data were collected using small m/z windows, which optimize sensitivity (Southam et al., 2007). Briefly, samples at a concentration of 0.014 mg dry mass mL−1 were analyzed using direct-infusion ESI on an LTQ FTICR hybrid linear quadrupole ion trap FTICR mass spectrometer (Thermo Finnigan). The ESI source was operated in positive mode with a spray voltage of 2.8 kV, a tube lens offset of 140 V, and a capillary temperature of 200°C. The instrument was calibrated using an automatic routine based on a standard calibration solution containing caffeine, peptide Met-Arg-Phe-Ala, and Ultramark 1621 (all products of Sigma-Aldrich). The 700 to 1,200 m/z range was divided into multiple 30 m/z selected ion monitoring intervals with 5 m/z overlaps at both ends. Each interval was scanned for 2 min (approximately 10 scans) using a selected ion monitoring MS target automated gain control value of 2 × 105 accumulated ions with a resolution of 500,000 at m/z 400. FTICR mass spectra from individual scans within a mass interval were averaged using Xcalibur 2.0 software, and peaks with average counts greater than 1,000 were exported to Excel 2010.
Statistical Analysis
Significance was determined at P < 0.05, after correcting for the false discovery rate, using Excel 2010. Comparisons were between stress samples and their controls.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Possible ox-lipid structures and their MS fragmentation.
Supplemental Figure S2. Bacterial counts per leaf dry mass.
Supplemental Figure S3. Ox-lipids from control and low-level treatments.
Supplemental Figure S4. Ox-MGDG, ox-acMGDG, and PA in wounding and bacterial infection.
Supplemental Figure S5. LPC, LPE, and PA under treatments.
Supplemental Table S1. QTOF MS data supporting Table 2 ox-lipid identifications.
Supplemental Table S2. FTICR MS data supporting Table 2 ox-lipid identifications.
Supplemental Table S3. Simplified designations of diacyl ox-lipids (1–59).
Supplemental Table S4. Simplified designations of acMGDGs (60–86).
Supplemental Table S5. All data: ox-lipids under treatments.
Supplemental Table S6. Averages and sds: ox-lipids under treatments.
Supplemental Table S7. All data: normal lipids under treatments.
Supplemental Table S8. Averages and sds: normal lipids under treatments.
We thank Gail Ragan for helpful suggestions. We are grateful to Mark Ungerer for generously allowing us to use his freezing chamber.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ruth Welti (welti{at}ksu.edu).
↵1 This work was supported by the National Science Foundation (Collaborative Research grant nos. MCB 0920663, 0920600, and 0920681 to R.W., J.S., and X.W., respectively). Equipment acquisition at the Kansas Lipidomics Research Center was funded by the National Science Foundation (grant nos. EPS 0236913 and DBI 0521587), the Kansas Technology Enterprise Corporation, the Kansas IDeA Networks of Biomedical Research Excellence of the National Institutes of Health (grant no. P20 RR16475), and Kansas State University. This is contribution no. 12–036–J from the Kansas Agricultural Experiment Station.
↵[OA] Open Access articles can be viewed online without a subscription.
↵[W] The online version of this article contains Web-only data.
- Received October 29, 2011.
- Accepted November 10, 2011.
- Published November 15, 2011.


text:
One of the keys to good health is knowledge. Through the resources offered here, we believe you can learn more about various conditions, their risk factors, and lifestyle changes you might want to make.
Nutrition Tip of the Week:
Our registered dieticians offer a new tip each week to help you make healthy food choices for you and your family. You can also see a list of past tips.
Click on the links below for lists of online articles written by our health providers, links to other web sites, and books we recommend on various health care topics.
- Children's Health
- Women's Health
- Men's Health
- Elderly/Geriatric Health
- Diet & Nutrition
- Hormone Replacement Concerns
- Other Illnesses/Diseases
- List All Resources
Disclaimer: This information is provided for educational purposes only. It should not replace a visit with your health care professional. Call your doctor if you need more information or have additional questions.


text:
Yelm Community Schools' Student Support department is planning its third move of the Supporting Independence Through Employment (SITE) House to a location closer to Yelm High School.
The SITE House is expecting to move to a new and larger location on the corner of Tahoma Boulevard and Tornado Alley by spring of next year. The district paid $350,000 for the 2-acre property, which includes a 3-room, 3-bath house currently under renovation. The property is currently undergoing $153,000 worth of improvements, which includes paving a driveway, landscaping and septic work.
The SITE House is an 18-21 year old program lead by Student Support, which assists special-needs students' transition from high school into the workforce. The program offers training and volunteer opportunities for students to gain applicable workforce skills.
SITE House teacher Nora Sutherland said she's looking forward to moving to a larger location, even though their current location on the 400 block of Yelm Avenue works as a great access point to downtown businesses.
"It'll be interesting to see the things we don't need and the things we do," she said.
Sutherland said she's excited about the extra space at the new location because it will allow the students to work different training stations at the same time, such as organizing clothes and books.
"Just the ability to have training stations that I don't have to take down…" Sutherland said.
The move to the new location could open up opportunities to serve more students, Sutherland said. The SITE House currently serves 12 students and has the resources to serve up to 16.
Superintendent Brian Wharton said the district has always held an interest in the property behind the high school. The property would offer the district options to build another school if needed, and the decision to bring the SITE House closer to the high school gives the program the ability to closely collaborate with the high school.
"There's a huge need in our community for programs like that," Wharton said. "I just think it was smart to look long term as to where the district needs to grow, and it makes sense to have our location back there."
Wharton said it would be in the district's best interest to sell the current property on Yelm Avenue following the move, although no concrete decision has yet to be made.


text:
Spring is the season when all allergies come at its peak for both your dogs and cats. Allergies are basically an overreaction of the immune system. The immune system will take in something that would generally be harmless and act as though it is bad. This can be dangerous to your pet's health.
Flea allergies: Some pets are very allergic to fleas. Just one single bite will trigger this bad reaction. They will chew and scratch the area that was bitten such as the tail. This will make their hair fall out, and their skin very sore and even raw.
Inhalant allergies: Your pets can be allergic to the "Spring scents" such as pollen, ragweed and so on. This type of reaction will make their skin itch, their eyes water and cause mucus buildup and sneezing. Pets will also scratch and chew on their skin.
There are also other allergies not related to the Spring season. Your pet may be allergic to food. These symptoms will usually be digestive symptoms; vomit or diarrhea. Signs of uncomfort along with scratching and biting are also common.
If your pet is scratching, chewing or constantly rubbing his body against the floor chances are it's an allergy related problem. The sooner we get them checked out, the sooner they will feel better.


text:
- Six new stations for the YUS line
- All new authentic stop announcements for all stations
- Route built with real tunnel length and ATC signal annimations
Free for all YUS 4.0 customers.
Free for all YUS 4.0 customers.
Hello! I want to talk about their native place. I was born in the settlement Vazyuk, it is in the Kirov region, Russia. My settlement (although it...Last Post By: Vazyuk Yesterday, 01:46 PM
Picking back-up where I left off a couple of years ago with some additions to the LS&I route: Jeff M.Last Post By: wooohooo Yesterday, 11:59 PM
Name of Route:PRR-Eastern-Region-v2 Version and Release Date: Version 2.0 11April2019 Description: This route is a...Last Post By: asteph627 Today, 12:51 AM
Kransoyarskaya (Krasnoyarsk's) railway locates in Kranoyarsk region, during my trip to Irkutsk in August 2017, I was go to sleep in Novosibirsk and...Last Post By: Vazyuk Yesterday, 01:51 PM
Classification yard Irkutsk-Sortirovochniy (Иркутск-Сортировочный) locates in western part of city Irkutsk. On this station starts bypass track only...Last Post By: Vazyuk Yesterday, 01:47 PM
I've been remiss at showing off what I've been working on. Time to fix that . . . I got inspired by Mark's activities for the Jersey Coast and at...Last Post By: conductorchris Yesterday, 06:53 PM
Various KCS trains in and around Kansas city.Last Post By: trainman_47 Yesterday, 07:53 PM
Really worthwhile freeware route. http://www.vpris.co.uk/ Info Free download from SteamSoundsSupreme. Mike.Last Post By: NorthernWarrior Today, 03:13 AM
Brought to my attention this morning that one of the ACL gondolas I made still uses the textures and road number from the other ACL gon I drew. The...Last Post By: charland Yesterday, 06:59 PM


text:
Northern Ireland Under-21s finished runners up in their UEFA U21 EURO Qualifying group following a 1-0 win over Slovakia, but narrowly missed out on a play-off place.
They needed a big win over their opponents and results elsewhere to go their way to stand any chance of making the Euro 2019 play-offs.
In the end Ian Baraclough's side secured a 1-0 win and leapfrogged Slovakia into second place thanks to a second half goal from Glenavon's Mark Sykes.
It has been a fantastic effort from Northern Ireland to finish the group just four points off Spain in top spot.
The manager has overseen six wins, two draws and two defeats in his 10 games in charge, with the highlight last month's 2-1 away win over Group Two leaders Spain.
The visitors had to play the entire second half with only ten men following the dismissal of Laszlo Benes who picked up his second yellow card of the game after a foul on Jamie McDonagh on 44 minutes.


text:
Dear Christel,
Your paintings keep getting better, richer, deeper. It is as if the deeper you allow yourself to go within, the more depth is lent to the canvas. Well done, I'm so proud when I see what you are doing now and knowing where you have come from. Love Dena


text:
Yogalehrerin - Vertretung (english)
Michaela has flirted with yoga and meditation since college, but not always consistently. Yoga would come in for a while, and then disappear as life got busy. Then life would get too busy and she would once again need yoga. Finally after doing this dance for many years, she discovered that if she committed to practicing yoga regularly and spending time being still, everything was lighter and more manageable.
This was in 2013, when Michaela was teaching acting and performance at an international school and also producing the annual school musical (which means working 7 days a week, 140%). Completely out of balance again, she found that her mind was so full that it was increasingly difficult to let go of the day and all the "to dos", difficult to be present in even the simplest of conversations, and impossible to sleep. A friend invited her to a yoga class she was teaching and Michaela discovered (remembered?) that when she practiced "letting it go' while on the mat, she could "let it go" easier off the mat. So she began having a daily practice - even if it was only 5 minutes! She started sleeping better, she was more engaged in daily life, the stress released on the mat and stayed there. She was completely convinced that this was the secret to balancing all the demands of life and absolutely needed to share this with the world!
As an acting teacher, bringing yoga into her school classroom came naturally. Her teenage students loved it, but she still noticed her colleagues and parents stressed, tired and bogged down. She wanted to share with them! So in 2016, she completed the Yoga Alliance certification under Angela Boscardini and Julie Martin and began that journey.
Michaela believes that bringing union (yoga) with movement, meditation and the breath is the key to maintaining a balanced life, discovering the happiness within, staying young at heart, in mind and in body, learning how to "let it go" and build confidence on and off the mat.
Her lessons are a balance of moving with the breath, challenging the body, exercising the mind, and igniting the fire in our hearts to live more fully on and off the mat. She loves discovering when something we practice on the mat translates to life outside the studio. This, to Michaela, is the power of yoga.


text:
Hidden Treasures
in Columbus, OH
(showing 1 - 3 out of 3)
If you're looking for must-see hidden spots in the area, you've come to the right place. We have everything you're looking for to enjoy the town – whether for a few, fun vacation days or as a new resident. The area is full of quaint little plazas and gardens, and gorgeous cathedrals for a fun-filled afternoon with friends and family.
Among hidden treasures, you will also find important memorials and historic buildings that you won't want to miss during your outing. Below you'll find a detailed description of each location, along with their address, phone number, and website, to check out more information. We also have a useful map to easily plan your visit to every spot you want to hit.
Franklin Park Conservatory & Botanical Gardens
1777 E Broad St
Columbus, OH 43203 Directions
Blooming enthusiasts, visit the Franklin Park Conservatory. Visitors of all ages grow happy with the sights, scents, and attractions in bloom across the 88-acre botanical garden in downtown Columbus. Fertilize your botanical interest when you wander through exotic plant collections, special horticultural exhibits, and attend educational work shops.
German Village
588 S 3rd St
Columbus, OH 43215 Directions
Shop, dine and stroll in historic German Village. Shop unique merchant wares like the city block- long store The Book Loft and Caterina for European housewares, arts and gifts. Dine at indie diners like Columbus Food Adventures. Stroll alongside colorful sidewalk merchants for fresh fruits and flowers and more.
Hayden Falls
4326 Hayden Run Rd
Dublin, OH 43017 Directions
Get misty at Hayden Falls. The falls are located in Griggs Reservoir Park on the west side of the Scioto River in Columbus. Travel down a stairway to the boardwalk near the base of the fall to experience its wild and scenic wonder waiting to tumble enjoyable misty moments for all.
Page Sponsor
Air Supply Heating & Air Conditioning, Inc.
- • Air Conditioning
- • Indoor Air Quality - 24 Hour Services
- • Licensed - Bonded - Insured - OH License # 36848


text:
My baby is now the size of a 5 pound bag of flour although she is still a skinny little thing -- fat has yet to settle in under her skin and fill out her frame. This week, her little hands are fully grown, complete with fingerprints and fingernails!
Come back each week to get an update on the size of my baby. I think it is cool to know what is going on in there so i thought i would share it with you all.
Also just to catch up till now, Our baby has hair, can hear and can tell if she is rightside up or not. I think there are more things but those are the main ones.