Multiple research collaborations with partners in India as diverse as the National Institute of Hydrology (NIH) and The Energy and Resources Institute (TERI) have brought new insights to the challenges that India faces.
In particular, IIASA applied systems analysis has brought a global perspective, interdisciplinary research expertise, and policy relevance to issues ranging from the future of India’s energy system to increasing the country’s food production.
The percentage of the world’s population that is above or below the ‘replacement level of fertility’ has long been used as a measure of demographic development. A new study revisited how this metric is calculated and how useful it really is in terms of informing policy decisions. More
There has been a substantial increase in food production over the last 50 years, but it has been accompanied by a narrowing in the diversity of cultivated crops. New research shows that diversifying crop production can make food supply more nutritious, reduce resource demand and greenhouse gas emissions, and enhance climate resilience without reducing calorie production or requiring more land. More
Two fundamental goals of humanity are to eradicate poverty and reduce climate change, and it is critical that the world knows whether achieving these goals will involve trade-offs. New IIASA research for the first time provides a basis to answer this question, including the tools needed to relate basic needs directly to resource use. More
Last edited: 19 March 2020
Info Sheet on IIASA Activities with India
Ghislandi S, Muttarak R , & Shaikh M (2020). Empowering girls, delaying marriage: Exploring the role of marital age and education on domestic violence in India. IIASA Working Paper. Laxenburg, Austria: WP-20-002
Diamond-Smith N, Saikia N , Bishai D, & Canudas-Romo V (2020). What has contributed to improvements in the child sex ratio in select districts of India? A decomposition of the sex ratio at birth and child mortality. Journal of Biosocial Science 52 (1): 27-36. DOI:10.1017/S0021932019000221.
Moradhvaj & Saikia N (2019). Gender Disparities in Health-care Expenditure (HCE) and Financing Strategies (HCFS) for In-patient Care in India. SSM - Population Health 9: e100372. DOI:10.1016/j.ssmph.2019.100372.
International Institute for Applied Systems Analysis (IIASA)
Schlossplatz 1, A-2361 Laxenburg, Austria
Phone: (+43 2236) 807 0 Fax:(+43 2236) 71 313


text:
Ice Skate Joy Blue & White Satin Ribbon 2.5" x 10 yards
Regular Price: $ 14.95
- Sent with love, from our shop in New Jersey
- In stock, ready to ship
- Inventory on the way
This white satin ribbon depicts grey and blue ice skates stuffed like stockings. The boots are decorated with snowflakes and filled with peppermints, candy canes and holly. A tag on the grey boot says, "Noel," while the one on the blue boot says, "Joy." White snowflakes dot the background and silver glitter makes the design's outlines sparkle.
Size: 2.5" W x 10 yards
Material: Satin, Glitter, Wire

domain: design


text:
1 2312 Tool Steel Material P20 Alloy Steel Mould Steel
1. Steel Grade:AISI P20 2. Product name:AISI P20 Alloy Mould Steel Plate width:1000-3000mm Length:1000-12000mm 4.Heat Treatment:as required 5.Standard:ASTM,GB,JIS,DINsp.info China DIN 1.2312 AISI P20+S/P21 JIS Nak80 Alloy Tool Steel DIN 1.2312 AISI P20+S/P21 JIS NAK80 Alloy Tool Steel for Plastic Mould 1:NAK80 steel introduction NAK80 steel is a precipitation or age-hardened mold steel with a uniform through hardness of approximately 40 HRC. It is super clean, Vacuum-Arc Remelted steel with uniform grain structure, uniform hardness, and excellent dimensional stability.sp.info What You Should Consider When Purchasing Modified P20 Nov 01, 2004 · They naturally gravitated to building molds with the chrome-moly steels they were used to, such as DIN 1.2311 and DIN 1.2312, or their close cousin, AISI P20. P20 differs from region to region in the worldwide market. Europeans generally use the DIN spec materials (1.2311 and 1.2312), while in Japan, PX5 is the new standard P20.sp.info Non-Stainless Steels – EDRO9 rows · Powder-metallurgical produced cold work tool steel with extremely high wear resistance,
P20 Steel Price, 2021 P20 Steel Price Manufacturers
Recommended product from this supplier. Die Steel 1.2311/ P20 Hot Rolled Steel Plate Plastic Mould Steel Flat. FOB Price:US $ 1300-1800 / Ton. Min. Order:2 Tons. Standard:DIN, JIS, AISI, ASTM, GB, BS. Technics:Hot Rolled. Application:Die Steel Bar. Surface:Polished.sp.info M200 - BÖHLER Edelstahl GmbH & Co KGCorrosion resistant and non magnetic steel; Ferritic and martensitic steels, incl. precipitaion hardening steel Hardenable corrosion resistant steels; Ni-Base alloys; High speed steels; Hot work tool steels; Creep resistant steels; Hire work; Hire rolling; Precision wire manufacturing DIN EN Others; 1.2312:40CrMnMoS8-6:AISI:~ P20 sp.info P20 AISI P20 Tool Steel - Tool Steel Specification and AISI SAE P-20 Tool Steel. AISI SAE P-20 is a pre hardened high tensile tool steel which offers ready machineability in the hardened and tempered condition, therefore does not require further heat treatment.This eliminates the risks, cost, and waiting time of heat treatment thus avoiding the associated possibility of distortion or even cracking.sp.info Tool Steel Supplier - West Yorkshire Steel - Only £25 UK Plastic mould steel specifications are held in International Standards such as BS4659, BS EN ISO 4957, AISI, Werkstoff Nr and DIN designations. Plastic mould tool steel grades offer a high wear resistance, good polishability and etching ability with adequate corrosion resistance.
Tool Steel- Hot Work - High Speed Plastic Mould Alloy
WELCOME TO VIRAT SPECIAL STEELS The Leading Company VIRAT SPECIAL STEELS PVT. LTD. is one of the largest Importer, Exporter and Stockist of Die Blocks (DIN 1.2714, DB6, H13, H11), Tool Steels, Die Steels, Special Steels and Alloy Steels in various grades.The company maintains biggest Steel Yards and hold a high level of inventory of Special Steels from the world’s leading sp.info China Material P20 1.2311, Material P20 1.2311 3Cr2Mo Steel, P20 Steel Material, 1.2311 /40crmnmo7/35cmd7. Min. Order:5 Tons. Standard:DIN, JIS, AISI, ASTM, GB, BS. Application:Mould Making. Surface:Black or Machined. Delivery Condition:Eaf or ESR. Shape:Round or Plate. Size:2-900mm. Dongguan Kasen Steel Co., Limited.sp.info AISI D3 tool steel(1) Material properties. High carbon high chromium alloy tool steel. The mass fraction of the steel carbon is 1.45% ~ 2.3%, the mass fraction of chromium is about 12%, and it contains a small amount of molybdenum and vanadium, which belongs to the high-carbon high-chromium minellite steel. The steel has more wear resistance, hardenability,sp.info DIN 1.2379 /AISI D2/JIS SKD11,DIN X155CrVMo121,high 1.2379 die steel is a kind of high carbon high chromium alloy tool steel. It has high hardness and wear resistance after heat treatment, and has strong hardenability and good dimensional stability. 1.2379 die steel and NAK80 die steel are suitable for making. High precision long life cold working die and thermosetting plastic mold. It equivalent steel grade:
p20 1.2311 steel, p20 1.2311 steel Suppliers and
Steel P20 1.2312 Cold Work Drawn Alloy Toll Stainless Steel HSS Steel Round Bars Best Quality Bar Round ASTM AISI P20 S / WNr DIN 1.2312 US $2.00-$4.00 / Kilogram 100.0 Kilograms (Min. Order)sp.info tool steel aisi p20, tool steel aisi p20 Suppliers and offers 2,580 tool steel aisi p20 products. A wide variety of tool steel aisi p20 options are available to you, such as standard, processing service, and grade.sp.info AISI M42 Steel - JIS SKH59 - DIN 1.3247 - Songshun SteelJul 31, 2019 · And it also is called JIS SKH59 and DIN 1.3247 steel. It is mainly used for high toughness precision wear-resistant hardware cold stamping die and cutting tools. It has high toughness and hardness, could reach to 70HRC. Its hot hardness is high and easy to grind and cut. The cutting tools which are made of M42 steel can cut cast superalloys, titanium alloys, and ultra-high-strength steels.sp.info Mat.No. 1.2311, DIN 40CrMnMo7, AISI Approx. P20 - Co., Ltd.Designation by Standards Mat. No. DIN EN AISI 1.2311 40CrMnMo7 - Approx. P20 Chemical Composition (in weight %) C Si Mn Cr Mo Ni V W Others 0.40 0.30 1.45 1.95 0.20 - - - - Description Prehardened plastic mould steel with good machinability, better than steel grade Mat. No. 1.2312, suitable for texturing.
PLASTIC MOULD STEELS
C Cr Mo Ni V Others DIN / EN AISI CORROSION RESISTANT STEELS BÖHLER M200 0.40 1.90 0.20 – – Mn = 1.50 S = 0.08 < 1.2312 > 40CrMn-MoS8-6 ~ P20 ¸ High wear resistance BÖHLER grade Corrosion resistance*) Wear resistance Toughness Polishability**) Machinability in as-suppliedsp.info Plactic mold steel/Die steel - HK JIMI STEEL CO., LTD Tool Steel, Die Steel, Saw Blade manufacturer / supplier in China, offering Precision Ground, Standard Size AISI D2 SKD11 High Chromium Steel Is Ideal for The Highest Wear Resistance Applications, Precision Ground Flat Steel Prefinished Tool Steel Flat Bar Alloy Steel D2 Cr12MOV SKD11, DIN 1.2379 Special Steel DIN 1.2379/SKD11/D2 Special Steel Retangular Square Bar and so on.sp.info Special Steel Manufacturer WALDUN SteelSpecial steel, is also called alloy steel which contains one or several alloying elements In the carbon steel.Special steel has a variety of special properties, such as strength, hardness, plasticity, toughness, wear resistance corrosion, and many other excellent performance.sp.info P20+S easy cutting plastic mold steel plate pre-hardness DIN 1.2312 steel is a high quality plastic mold steel material. Steel 1.2312 is material produced by add about 0.08% sulphur on material 1.2311. The S element improves free cutting ability of 1.2311 steel . 2312 material is usually delivered in pre-hardness condition and hardness 28~34HRC. ASTM P20+S steel is equivalent to DIN 1.2312 material.
20NiCrMo2-2 Alloy Steel DIN 1.6523 AISI 8620 steel
DIN 1.6523 steel also improves the content of Manganese, so it has better hardenability and strength than 18NiCr5-4. 20NiCrMo2-2 is suitable for parts which require good toughness and wear resistance. The nickel content contributes to case toughness at high strength with above average core toughness.sp.info What is tool steel? - Special steel china supplier-OTAI ②Alloy tool steel. Alloy tool steel is a special quality Medium carbon or High carbon alloy steel( Carbon content >0.25%). Alloy tool steel’s Quenching, quenching, wear resistance and toughness all higher than carbon tool steel. According to the purpose can be roughly divided into cutting steel, mould steel and measuring tool steel 3 classes.sp.info DIN 1.2083 / AISI 420 / GB4Cr13 Plastic Mold Steel DIN 1.2083 Plastic Mold Steel is a kind of Martensite stainless steel, with good machincability and excellent corrosion resistance, polishability, and high strength and wear resistance after heat treatment. It is widely used for making plastic moulds and transparent plastic molds which require high load, wear resistance, and work under the sp.info

domain: chemistry


text:
I have added the third route page on the blog covering details of our trip from Italy through Croatia, brief visit to Bosnia Herzegovina (BiH), and back to Italy.
You will need to visit the blog to read the detail if you’re interested. For non-bloggers, click here and then on the Route page in the top right hand corner. Then click on underlined links (Croatia and BiH) for the detail.
The Croatian Adriatic coast is a great place for a ‘beach holiday’. It is geared up for tourism, mainly for German tourists of which there are many. You generally get greeted in German, but most people speak some English (and Italian). It has beautiful calm, clear water, great for swimming in. Although most people spend their time grilling themselves on the shore and only going into the sea for a cooling paddle. It’s a bit like Salthill on steroids (the Irish will know what I mean!) with great weather.
A lot of the coastal towns are beautiful (Dubrovnik, Split) but very crowded. They get a lot of Cruise ships.
You do get an underlying feeling that Croatia has sold its soul to the tourist. Great coast for the tourist less good for ‘the traveller’.
It’s worth going inland for a break and to get away from tourist pricing! It’s more mountainous, fewer people, more ‘real’.
BiH is even more so, mountainous, outdoor sports, poorer, less developed, hard to sum up given we only had 2 days, one night there … but it was certainly memorable.
Just checked the last words of the last diary blog – something about never forgetting our one night in Bosnia-Hercegovina. Prophetic words – there was an earthquake later that night. We both woke around 1am to a loud rumble and the van shaking. Didn’t last long. In the morning Sandra (campsite owner) told us it measured 4.1 on the richter scale, and the epicentre was pretty nearby. It’s the first earthquake we’ve experienced.
We drove up into the mountains, via the Catholic pilgrimage site of Medugorje, and on to the Kravica waterfalls. These are not as spectacular as the Plitvice Lakes, but have the huge advantage that you can swim in the pool beneath them. It was cool, refreshing and totally delicious swimming there. We stayed a while, had lunch, and then drove on, out of Bosnia-Hercegovina by a very scenic and interesting route.
If you look at a map you’ll see there’s a bit of Bosnia that goes down to the coast, splitting Croatia in two. After having had a bit of trouble entering Bosnia we did not want to leave it then have to re-enter it to get through to Dubrovnik, so we planned a route that led down that land corridor to the coast, then out of Bosnia. Clearly though it’s not a well-used route. The road surface was worthy of Sicily and it was single track with sporadic passing places. Didn’t see much other traffic thankfully!
Back in Croatia, on good roads, we headed towards Dubrovnik and stopped at a campsite about 16km north of the city.
Dubrovnik’s been high on our list of must-see places. I was fearful that after so many gorgeous medieval walled towns it would be just another such, but no – it’s in a league of its own and well worth a visit. Despite extensive damage in the 1990s wars, it’s been fully restored. The walls are complete and you can walk around the top of them, and the streets are very pretty. Several areas around the city have been used as locations for filming Game of Thrones, and we tried to find some of these. Visited a few museums, had a very nice lunch out.
The day was very warm and humid which made the visit less comfortable than I’d have liked but we really enjoyed it. Took a bus from outside the campsite to get there and back which was a good no-hassle way of doing it!
A day of future planning. As we’re heading into Montenegro and that’s outside Vodafone’s roam-free area, we thought we should find campsites, plan a route etc while we still have access to the internet (it’ll be £3 a day for pretty limited data allowance in Montenegro and I’ll want to save that for Google maps!) So we spent a lot of the day online, planning. I also did some writing.
Down a steep hill from the campsite is a small beach and cafe, so we had a swim and lunch there. Very refreshing, but walking back up the hill made us too hot again!
We had planned to move on today to Montenegro but we rather like this campsite so decided to spend one more day here. It’s lunchtime as I write this, and if all goes to plan it’ll be the last blog entry until we’re back in Italy in about a week, after taking the ferry from Bar to Bari.
That campsite outside Split was truly lovely. Everything (except perhaps the shower heads if I’m being picky) was spot on. We spent the first day there lounging around, catching up on chores and generally relaxing, but also had a swim in the sea and breakfast at the campsite restaurant.
Cycled into Split. First time on the bike since my fall in Zadar and I’ll admit I had lost some confidence. It was about 8km from the campsite to Split town centre, along a country road with some hills to start with (and we got chased by a dog which didn’t help!) and then the route was along a beach front road before entering the city.
We locked up the bikes and went around the old parts of the city on foot as usual. Split’s interesting in that the old Roman retirement palace built by Emporer Diocletian is still in use – filled with shops, restaurants and businesses, obviously parts of the buildings are more modern but all use parts of the original Roman walls etc. Fascinating place to wander around.
Cycled back to the campsite at Strobec via a slightly different route which avoided the dog. And then straight in the sea to cool off – it was a really hot day. Temperatures have been creeping up and up and are now around 30 during the day. We ate the evening meal at the campsite restaurant.
Another day of lazing around the campsite. I have some writing edits to get on with so I do need the time! But of course we did go in the sea again.
Time to move on. After calling at a shopping mall for some food we drove via the coast road then through the mountains, into Bosnia-Hercegovina. At the border we had to show passports then were asked for our ‘green card’ – something to prove we had insurance. Hmm. We don’t have one. We showed our insurance certificate. Lots of discussion amongst the border officials (who were holding our passports) then one said perhaps we give him something ‘for coffee’. Ignatius realised the lay of the land, said well we don’t have to go into Bosnia, perhaps we’ll just go back to Croatia, and at that the official decided we could progress after all.
We drove on to Mostar. Huge difference between coastal Croatia (very busy, touristy, a little bit ‘fake’) and Hercegovina (the southern part of Bosnia-Hercegovina). Here you can tell it’s much poorer, less tourist money coming in, still scarred by the 1990s wars. It reminded me in parts of Sicily, whereas the rich Croatian coast is more like the south of France only full of Germans.
At Mostar we paid an arm and a leg to park near the Stari Most (Old Bridge – the one that was built in the 16th century, blown up by the Croats in 1991 then reconstructed in 2004). Wandered through the old town on slippery cobbles, over the bridge, had a look in a museum which screened a video about the rebuilding of the bridge, and generally sweltered under the unforgiving sun.
Headed out of town southwards where we found a lovely little campsite by the river. The woman (Sandra) who runs it is very friendly, offered us beers on the house. We chatted to her for a long time. She has an Irish friend and on hearing Ignatius is Irish came out with ‘céad míle fáilte’ which was not a phrase we expected to hear in Bosnia-Hercegovina. She used to work at Medugorje – a Catholic pilgrimage site where the Virgin Mary appeared to a bunch of children back in 1981 – and on hearing Ignatius’s mum has been there insisted on giving us a little gift to pass on when we come home. I’ve rarely known a welcome as friendly as this at any campsite. This is likely to be our only night in this country but it’s not one I’ll forget.

domain: geography


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ACTION Stations was a week of activities laid on for young people with disabilities at Sayers Croft Field Centre in Ewhurst.
With the help of funding from the Duvollet Trust, the centre gave up to 12 young people a day the chance to participate in a range of activities last week.
For the more adventurous, there was climbing, ropes courses, raft building and archery, while for those who prefer a quieter life there was kite making, clay art, nature discovery walks and a treasure hunt in the nature reserve.
Sayers Croft community education officer Ellie Rickman said: “The grant enabled us to run a packed programme with lots of staff on hand to give extra assistance to those who needed it.
“The children were able to try things they had never tried before in a safe and friendly environment,” she continued, adding: “The week went really well and everyone had a great time.”

domain: sociology


Etihad Rail, the developer and operator of the UAE national railway network, has signed a 1.2-billion-dirham ($327 million) agreement with Spain’s CAF Group for designing, manufacturing, supplying and maintaining passenger trains.
The agreement was signed at the construction site of Etihad Rail’s first passenger train station being built at Sakamkam in the heart of Fujairah city, the state-owned company said in a press statement.
CAF will design, manufacture, supply, and provide maintenance for passenger trains as per the European standards, the statement said, adding that each train will have a seating capacity of over 400 passengers, and will run up to 200 km/hour.
The development of a passenger railway, which will connect 11 cities and regions in the UAE, is one of the three strategic projects of the National Railways Programme.
Etihad Rail said the passenger railway network is expected to cut travelling times by 30-40 percent compared to other modes of transport, with Abu Dhabi-Dubai, and Dubai-Fujairah segments expected to take 50 minutes each, while travelling from Abu Dhabi-Al Ruwais stretch will take 70 minutes.
The number of passengers on Etihad Rail is expected to touch more than 36.5 million annually by 2030.
In February 2022, Etihad Rail had signed an agreement with First Abu Dhabi Bank (FAB) for financing the passenger transport services with a total value of 1.99 billion ($542 million).


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Don’t miss the annual end of summer BBQ with Friends of Fire Mountain!
On August 20th at 4:00 p.m. join your neighbors at the cul-de-sac at the end of Avocado on Dunstan.
You’ll see flyers posted in the neighborhood as reminders to bring a little something for the grill, a dish to share, something to drink, something to sit on and a canned/boxed food item for the food drive.
Kids, dogs, and visiting friends and relatives are welcome!
FOFM will provide the plates, forks, napkins, cups and of course, the grills.

domain: food


text:
SAN JUAN, PUERTO RICO — Police revealed Saturday that a caller told them a bomb was in the Dupont Plaza Hotel only hours before the New Year`s Eve blaze that killed at least 95 people.
San Juan police spokesman Jose Lopez told reporters Saturday that police had received a phone call about noon Wednesday ``saying there was a bomb at the hotel.``
Two police officers went to the hotel, Lopez said, but left after security guards told them there was no problem.
The caller identified himself only as ``Santiago,`` Lopez said.
A spokesman for the hotel, Rissig Licha, said he did not know if the hotel`s security guards spoke with the police about the bomb threat on Wednesday or if they checked with management before sending the police officers away. ``We`re looking into it,`` he said.
He added that if the guards had spoken with the officers, ``They should have notified the management.``
Hotel director Bruce Shulman said he had no information about the meeting between police and hotel security guards concerning the bomb threat.
Earlier, he had said 30 guards were added as a precaution after three smaller fires broke out in the hotel within 10 days before the New Year`s Eve blaze.
A report of a separate warning was published on the front page of Saturday morning`s issue of the San Juan Star. The newspaper quoted ``a reliable source`` in the island government as saying three men had told the Special Investigations Bureau of the commonwealth`s Justice Department that an official of a union told them Wednesday to ``get out`` of the hotel casino
``right now because things are going to happen any minute now.``
The article said the official added: ``We are either going to close this hotel down or some people are going to get killed.`` The article did not name the three men but said they received the warning ``about 10 or 15 minutes``
before the fire.
Asked about the Star`s report, Jose Cadiz, secretary-treasurer of Teamsters Local 901 representing 225 hotel employees threatening to strike at midnight New Year`s Eve, said the union leadership knew nothing about plans to bomb or set fire to the hotel or about any prior warnings. In heated contract negotiations, he said, ``people do things, say things, make threats on their own.``
The arson probe, involving federal agents of the FBI and Treasury Department as well as commonwealth and San Juan police officials, has prompted a flurry of charges and countercharges.
Cadiz said his members told him the fire may have been caused by a lot of gasoline cans the hotel had stored in a hot kitchen area.
Attorney Guerry Thornton Jr. of Atlanta, who said he represented nine hotel customers, claimed that he has a witness` testimony that ``he was the last tourist to leave the casino before a hotel employee locked the doors, trapping inside many who were then killed by a fireball.``
Such an action justifies a lawsuit against the hotel, he said, ``because it indicates gross negligence and disregard for safety of guests.``
Shulman denied Cadiz`s and Thornton`s charges. He said: ``Guests who left the casino told me the doors were not closed or locked during the fire.``
He said the hotel does not have a policy of locking casino doors during any disturbance that might be a diversion for robbery.
Shulman did say hotel executive staff members had received threatening letters, which were turned over to police. The mail was anonymous, he said, and did not mention bombs.
Miguel Domenech, Puerto Rico`s director of tourism, is worried about speculative aspects of the investigation. ``Unfair publicity about rumors and hearsay could be very bad for tourism,`` he said.
After manufacturing and construction, tourism is Puerto Rico`s largest industry, he said, employing 55,000 people.
After a prolonged slump, which by 1984 saw Puerto Rico with 100,000 fewer cruise ship passengers, 215,000 fewer hotel guests and 3,000 fewer hotel rooms than five years earlier, tourism had rebounded to nearly its 1979 level, ``one of our best years,`` Domenech said.
The year that ended June 30 brought $739 million in tourist revenue. This year`s projected $770 million to $800 million might not be reached because of the Dupont Plaza tragedy, Domenech conceded.
``I haven`t seen reports of cancellations yet, but we`re concerned, and strategy meetings are being planned to see that this isolated incident doesn`t look like something that happens all the time here,`` he said.
Gov. Rafael Hernandez Colon, seemingly with an eye toward tourism, told a press conference Friday that the commonwealth has one of the best fire safety records, lowest fire casualty losses and steadiest insurance rates in the U.S. Conceding that the Dupont Plaza and other hotels don`t have sprinkler systems throughout, Hernandez Colon said a revised building code would require them in all hotels.
Although Domenech is worried about the future effects of the fire, on New Year`s Eve he was just another volunteer trying to help frightened people escape and find shelter elsewhere.
``We forgot about time. There was no countdown to the end of 1986,`` he said.
``In the distance, we heard firecrackers and some shouts. We thought that must have signalled the new year.
``Some of us embraced, and shook hands. A civil defense worker said he appreciated everyone`s help. We had a moment of prayer. It was very solemn.``

domain: news


text:
Brian and Judy Loberg, of Buffalo, will celebrate their 50th Wedding Anniversary on Friday, September 23, 2022. A private gathering with family and friends will be held in their honor.
Congratulations and love from their children, Becky (Jack), Cassie (Ryan), Jed (Jarilyn), and Neil, and their eight grandchildren.
Above, the couple is pictured on their wedding day; below, as they appear today. (P22, D25)
Wichita State Univ., KS
Allyson R. Arthur, of Monticello, graduated Magna Cum Laude with a BA degree in Communication Sciences and Disorders following the summer 2022 semester.
Univ. of Findlay, OH
Rachel Reed, of Delano, has enrolled at the University of Findlay for fall 2022. New students began their studies at UF on Aug. 15.
Reed is pursuing a degree in equestrian studies and a degree in equine business management.
BHS Class of 1950
The Buffalo High School Class of 1950 will be celebrating their 72nd Class Reunion on Thursday, October 6, 2022, at Huikko’s Bowling & Entertainment Center, 1207 Highway 25 North, in Buffalo.
Social Hour begins at 11:00 a.m. Lunch to follow. If you need more information, please call Dwayne Puffer at 612-867-2539 (cell) or 763-682-2381 (home), or just come. (P22, 29)
BHS Class of 1971
The Buffalo High School Class of 1971 will celebrate their 51st Class Reunion on Thursday, September 29, 2022, at Huikko’s Bowling & Event Center, 1207 Hwy. 25 N in Buffalo.
Social hour starts at 11:00 a.m., followed by lunch at noon. Spread the word.
For questions or more information call Chris at 763-360-4355. (P22)


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Destination Wedding – Bali
Not only is Bali a popular tourist destination amongst locals and foreigners alike, with its gorgeous sceneries and luxurious villas, it’s often the dream destination for couples to hold their dream weddings.
Many have chosen Bali as the place they want to tie their knot, to exchange their sacred vows, to share their special moment with their closest friends and families – and that’s a choice unlikely to be regretted.
MY wedding planner would be more than happy to help you plan your dream wedding in this breath-taking island of Bali, situated just 3 hours away from Kuala Lumpur International Airport by flight.
Write to us at [email protected] today for more information.

domain: travel


--- How To Order -----
1-) Choose your print size.
2-) Click add to cart.
3-) Click "Proceed to check out".
4-) When you check out, you can upload art add a note to seller for any request.
5-) Upload the files at check out or email to firstname.lastname@example.org
6-) If the background of the image is not transparent, please add "Remove Background" to cart and checkout together with the order!
- You can make your original T-shirt by sending us your image! Send us your design and we will have it ready for press.
- Can be applied to all types of fabric
- Can be applied to fabric of any color
- Better than vinyl, screen printing, DTG
- Full color + white
- Soft feel
- Great durability
- Gang sheets are welcome
- No minimum
- Fast turnaround
INSTRUCTIONS FOR HEAT PRESS
1. Preheat heat press on medium to hottest setting. Temperatures should be 330°F and pressure should be 70 psi and over
2. Iron on the garment to remove any creases and heat the fabric.
3. Place transfer face up in position on garment and cover with grease proof sheet. Never heat press directly on the Transfer.
4. Heat press for 15 Seconds.
5. Please let the transfer cool for 15 seconds before removing the tape.
6.After it gets cold, Peel the backing away from one corner to ensure transfers have stuck, if so peel remaining slowly. If the transfer has not stuck, repress for 10 seconds in a burst of 5 seconds to keep checking.
7. Once you have peeled away the clear film tape, place the grease proof sheet again and reput the garment in the heat press for another 5 seconds to fix the transfer in place.
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Our company started in March of 2002, and it was established with a goal to make super discount pricing for shower doors available to the general public. Over the years, we have grown steadily, becoming one of Metro Atlanta's premiere shower door companies specializing in heavy glass frameless enclosures.
Types of Shower Enclosures We Work On
Shower doors and enclosures are broken down into several "types" of systems that are determined by the amount of glass and metal components. These are frameless, semi-frameless, and framed.
An enclosure that uses the least amount of metal is called a frameless type. It uses thicker glass, ranging from 3/8" to 1/2", which can “stand up” without a frame. However, it will still have metal hinges, metal wall and glass connectors, and a metal handle.
Semi-frameless enclosures use 1/4" to 3/16" thick glass and have metal on only two sides of the door. However, this type must be closed into a framed system. This kind of frame is nearly the same as those used in framed shower enclosures.
Framed enclosures used the least glass and most metal amongst the three types. These systems generally use 3/16" glass and are “glazed” with rubber on all sides so that the glass touches the metal frame.
We focus on heavy glass frameless enclosures, offering the largest discounts for this type. Our crew uses “beveled” hinges and oversized 8” handles, cut glass out of square sheets, and mount panels using mini glass clips that are all color matched. Unless you request it, we will not use u-channels from mounting panels.
Beveled Edges Only
Only beveled edges are used for all glass-to-glass corners—no butted glass connections allowed. This method costs a little bit more, but it is the right way for enclosures. It also results in the most beautiful shower doors.
For our discounted shower door services, we accept cash, checks, all major credit cards, and payments through Zelle. Talk to us for any inquiries about these methods.
For all active and retired members of the military, first responders, and educators, we proudly offer you an additional 5% discount on our already low prices. Thank you for your service to our communities, our state, and our country.


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37 years after the Lutz family fled their Amityville house, director Eric Walter had the incredible chance to interview Daniel Lutz, a family member who was left traumatized after the unexplained events that happened when he was eight years old, and his experience has clearly taken a toll on him to this very day. For the uninitiated My Amityville Horror proves to be an intriguing and concise character study.
There’s a long and rocky history of TV producers trying to successfully adapt Stephen King novels. Could a new series capture the horror master’s sensibility while having nothing to do with his actual work? How 666 Park Ave. will be like The Shining.
Most people today bitch and moan about the younger generation. They claim that they are lazy, selfish, and don’t have any respect for anything. Obviously these people have never met Todd Crawford. Todd is only eighteen but he has already published several novels and short stories, and is a walking encyclopedia of all things horror related. Interview: Author Todd Crawford…
Inspiration goes full circle this summer at Whitby Abbey as the ruin which inspired a Gothic masterpiece, Dracula, plays host to the famous story this summer, when Dracula comes to the historic site! Centenary celebrations for Stoker’s legend at Whitby Abbey…
You won’t find lyrical passages of Stravinsky in Padraig Reynolds’s Rites of Spring, just the dissonant sounds of kidnapping, murder and human sacrifice. You also won’t find much new brought to the movable blood feast that is the horror movie, except a mildly novel juxtaposition of narratives. Rites of Spring, Horror From Padraig Reynolds…
John F.D. Taff can trace his obsession with horror to the nights he and his siblings would stay up waiting for their father to arrive home. “My dad worked as a St. Louis policeman and logged late and long hours,” recalls Taff. “My mom was a big fan of horror films back then, only she was too scared to watch them alone. She’d enlist my brother, sister and me to watch them with her. We all grew up loving those movies.” Meet John Taff; St. Louis Horror Writer with “Weakest Stomach Imaginable”
Horror screenwriter Mark Wheaton will soon add “published novelist” to his resume. Los Angeles-based ZOVA Books has signed a multibook deal with Wheaton for a young-adult series titled Finders & Keepers. Friday the 13th Writer Moving to Young-Adult Books…
Friday sees the worldwide cinema release of the Total Recall remake based on a short story by Philip K. Dick (here, the movie will open on Aug 2). Last month, on June 5, much-celebrated author Ray Bradbury passed away at age 91. So, this is a good time as any to remember for you wholesale the contributions of Chicago-born Dick (1928-1982) and Waukegan-born Bradbury (1920-2012) to dystopian science fiction.
Dale Zawada is a screenwriter and stand-up comic from Chicago. His first film was an award-winning romantic-comedy entitled Worth the Weight. Why do we care, you ask? Because Dale wants his next project to be horrific. Come meet Dale
- Limbus, Inc. Limited Edition Available For Preorder - February 14, 2013
- HWA 2012 Specialty Press Award Goes to Centipede Press - February 14, 2013
- Deeply Odd (Odd Thomas) - February 14, 2013
- The Vampires of Bloody Island - February 14, 2013
- Deep Cuts Anthology Honors Women In Horror - February 13, 2013
- The Demonologist: A Novel - February 13, 2013
- Dark Regions Press New Sponsor For Bram Stoker Awards® Weekend 2013 - February 13, 2013
- Chasm - February 13, 2013
- The Mad Scientist’s Guide to World Domination - February 13, 2013
- Ghostman - February 12, 2013

domain: literature


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Book your Reiki session now!
Looking for a soothing and revitalizing experience to restore balance to your mind, body, and spirit? Look no further than Reiki The Boundless Energy. As a leading practitioner in the field of alternative and natural medicine, we offer professional Reiki sessions that harness the power of universal life force energy to promote healing and overall wellness.
Why Choose Reiki?
Reiki is a gentle, non-invasive technique that originated in Japan. It is based on the belief that an unseen life force energy flows through all living beings, which can become disrupted due to various physical, emotional, and mental factors. By channeling this energy through the practitioner's hands, Reiki aims to remove energy blockages and restore harmony within the recipient.
At Reiki The Boundless Energy, our highly skilled practitioners have undergone extensive training and are dedicated to providing you with a deeply relaxing and transformational experience. With our customized approach, we tailor each session to address your unique needs and goals.
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The benefits of Reiki extend beyond just relaxation. Regular Reiki sessions can potentially:
- Reduce stress and anxiety
- Relieve pain and promote faster healing
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Whether you're seeking relief from a specific ailment or simply looking to enhance your overall well-being, Reiki can offer profound benefits on multiple levels.
How to Book
Booking your Reiki session with Reiki The Boundless Energy is quick and easy. Follow these simple steps:
- Visit our website reiki-boundlessenergy.com.
- Navigate to the "Booking" page.
- Choose your preferred date and time slot from the available options.
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Once your booking is confirmed, our friendly staff will reach out to you with all the necessary details and answer any questions you may have.
Experience the Healing Power of Reiki with Reiki The Boundless Energy
Don't miss the opportunity to embark on a transformative journey towards holistic well-being. Book your Reiki session with Reiki The Boundless Energy today and experience the profound benefits of this ancient healing practice.
Let us guide you towards achieving balance, harmony, and renewed vitality in your life. Get started on your path to wellness now!

domain: health


text:
Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host.
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.
21 Related JoVE Articles!
Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin
Institutions: University of Birmingham, University of Edinburgh.
The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (Bénézech et al.14
). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+
embryonic adipose tissue and make a LN/ EYFP+
fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+
fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.
Immunology, Issue 82, Adipose Tissue, Mesenchymal Stromal Cells, Immune System, Lymphoid Tissue, Lymph Nodes, Lymph node development, lymph node stromal cells, lymph node transplantation, immune responses, adipose tissue, adipose tissue stromal cells, stem cells
A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders
Institutions: University of California, Davis, Shriners Hospitals for Children - Northern California, University of California, Davis.
Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro
from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo
and in vitro
techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo
. Similarly, in vitro
culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.
Medicine, Issue 88, osteoclast, arthritis, minicircle DNA, macrophages, cell culture, hydrodynamic delivery
Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis
Institutions: NYU Hospital for Joint Diseases, New York University Medical Center.
Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules in the pathogenesis of osteoarthritis (OA) in vivo
. However, the detailed, especially the visualized protocol for establishing this complicated model in mice, is not available. Herein we took advantage of wildtype and progranulin (PGRN)-/- mice as examples to introduce a protocol for inducing DMM model in mice, and compared the onset of OA following establishment of this surgically induced model. The operations performed on mice were either sham operation, which just opened joint capsule, or DMM operation, which cut the menisco-tibial ligament and caused destabilization of medial meniscus. Osteoarthritis severity was evaluated using histological assay (e.g.
Safranin O staining), expressions of OA-associated genes, degradation of cartilage extracellular matrix molecules, and osteophyte formation. DMM operation successfully induced OA initiation and progression in both wildtype and PGRN-/- mice, and loss of PGNR growth factor led to a more severe OA phenotype in this surgically induced model.
Bioengineering, Issue 84, Mouse, Cartilage, Surgery, Osteoarthritis, degenerative arthritis, progranulin, destabilization of medial meniscus (DMM)
A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions
Institutions: Torrey Pines Institute for Molecular Studies, Cascade LifeSciences Inc..
Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro
technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2
tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo
behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.
Bioengineering, Issue 77, Cellular Biology, Biophysics, Physiology, Molecular Biology, Biomedical Engineering, Immunology, Cells, Biological Factors, Equipment and Supplies, Cell Physiological Phenomena, Natural Science Disciplines, Life Sciences (General), circulating cells, extravasation, physiological shear stress, endothelial cells, microenvironment, chemokine gradient, flow, chamber, cell culture, assay
Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies
Institutions: University of Oxford.
Kindlins are essential coactivators, with talin, of the cell surface receptors integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin β-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3's role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.
Virology, Issue 85, Heterologous protein expression, insect cells, Spodoptera frugiperda, baculovirus, protein purification, kindlin, cell adhesion
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets
Institutions: University of Alabama at Birmingham.
Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging. Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis. This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry. An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.
Immunology, Issue 85, bioenergetics, translational, mitochondria, oxidative stress, reserve capacity, leukocytes
Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy
Institutions: École Polytechnique Fédérale de Lausanne, Oregon Health & Science University.
Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.
Bioengineering, Issue 86, Intravital imaging, epifluorescence, two-photon imaging, Tumor matrix, Matrix remodeling
A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites
Institutions: Boston University School of Medicine, Boston University School of Medicine.
Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis
, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis
accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.
Immunology, Issue 90,
Pathogen-Induced Chronic Inflammation; Porphyromonas gingivalis; Oral Bone Loss; Periodontal Disease; Atherosclerosis; Chronic Inflammation; Host-Pathogen Interaction; microCT; MRI
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (https://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP
Institutions: West Virginia University , University of Pittsburgh, WVNano Initiative, Mary Babb Randolph Cancer Center.
Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro
antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro
antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus,
Group A Streptococcus
, and Neisseria gonorrhoeae
. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.
Infection, Issue 74, Infectious Diseases, Immunology, Microbiology, Medicine, Cellular Biology, Molecular Biology, Bacterial Infections and Mycoses, Musculoskeletal Diseases, Biological Factors, Platelet-rich plasma, bacterial infection, antimicrobial, kill curve assay, Staphylococcus aureus, clinical isolate, blood, cells, clinical techniques
Reverse Total Shoulder Arthroplasty
Institutions: Case Western Reserve University.
Reverse total shoulder arthroplasty was initially approved for use in rotator cuff arthropathy and well as chronic pseudoparalysis without arthritis in patients who were not appropriate for tendon transfer reconstructions. Traditional surgical options for these patients were limited and functional results were sub-optimal and at times catastrophic. The use of reverse shoulder arthroplasty has been found to effectively restore these patients function and relieve symptoms associated with their disease. The procedure can be done through two approaches, the deltopectoral or the superolateral. Complication rates associated with the use of the prosthesis have ranged from 8-60% with more recent reports trending lower as experienced is gained. Salvage options for a failed reverse shoulder prosthesis are limited and often have significant associated disability. Indications for the use of this prosthesis continue to be evaluated including its use for revision arthroplasty, proximal humeral fracture and tumor. Careful patient selection is essential because of the significant risks associated with the procedure.
Medicine, Issue 53, Reverse, Total, Shoulder, Arthroplasty, Rotator Cuff, Arthropathy, Arthritis, Glenoid, Humerus, Fracture
Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction
Institutions: San Diego Regenerative Medicine Institute, Xcelthera, Harvard Medical School, Division of SCI Research, VA Boston Healthcare System, Sanford-Burnham Medical Research Institute, La Jolla IVF.
There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3
. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3
. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7
. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10
. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13
. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo
derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14
(please see a schematic in Fig. 1
). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14
. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15
. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2
). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.
Neuroscience, Issue 56, stem cell, human embryonic stem cell, human, neuronal progenitor, neuron, human pluripotent cell, neuronal differentiation, small molecule induction, cell culture, cell therapy
A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid
Institutions: Tufts University School of Medicine, Tufts Medical Center.
Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo
; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2
. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6
. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering.
In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8
. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10
. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20%) 11-25
. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28
. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100%) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30
that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.
Cellular Biology, Issue 59, Chondrocytes, articular, human, synovial fluid, alginate bead, 3D culture
Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis
Institutions: Veterans Administration Medical Center, Memphis, TN, University of Tennessee Health Science Center, Memphis, TN, University of Tennessee Health Science Center, Memphis, TN.
Antibodies provide the ability to gain novel insight into various events taking place in living systems. The ability to produce highly specific antibodies to target proteins has allowed for very precise biological questions to be addressed. Importantly, antibodies have been implicated in the pathogenesis of a number of human diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multiple sclerosis (MS) and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP) 1-9
. How antibodies cause disease is an area of ongoing investigation, and data suggests that interactions between antibodies and various intracellular molecules results in inflammation, altered cellular messaging, and apoptosis 10
. It has been shown that patients with MS and HAM/TSP produce autoantibodies to the intracellular RNA binding protein heterogeneous ribonuclear protein A1 (hnRNP A1) 3, 5-7, 9, 11
. Recent data indicate that antibodies to both intra-neuronal and surface antigens are pathogenic 3, 5-9, 11
. Thus, a procedure that allows for the study of intracellular antibody:protein interactions would lend great insight into disease pathogenesis.
Genes are commonly transfected into primary cells and cell lines in culture, however transfection of antibodies into cells has been hindered by alteration of antibody structure or poor transfection efficiency 12
. Other methods of transfection include antibody transfection based on cationic liposomes (consisting of DOTAP/DOPE) and polyethylenimines (PEI); both of which resulted in a ten-fold decrease in antibody transfection compared to controls 12
. The method performed in our study is similar to cationic lipid-mediated methods and uses a lipid-based mechanism to form non-covalent complexes with the antibodies through electrostatic and hydrophobic interactions 13
. We utilized Ab-DeliverIN reagent, which is a lipid formulation capable of capturing antibodies through non-covalent electrostatic and hydrophobic interactions and delivering them inside cells. Thus chemical and genetic couplings are not necessary for delivery of functional antibodies into living cells. This method has enabled us to perform various antibody tracing and protein localization experiments, as well as the analyses of the molecular consequences of intracellular antibody:protein interactions 9
In this protocol, we will show how to transfect antibodies into neurons rapidly, reproducibly and with a high degree of transfection efficiency. As an example, we will use anti-hnRNP A1 and anti-IgG antibodies. For easy quantification of transfection efficiency we used anti-hnRNP A1 antibodies labelled with Atto-550-NHS and FITC-labeled IgG. Atto550 NHS is a new label with high molecular absorbtion and quantum yield. Excitation source and fluorescent filters for Atto550 are similar to Cy3 (Ex. 556 Em. 578). In addition, Atto550 has high photostability. FITC-labeled IgG were used as a control to show that this method is versatile and not dye dependent. This approach and the data that is generated will assist in understanding of the role that antibodies to intracellular target antigens might play in the pathogenesis of human diseases.
Neuroscience, Issue 67, Medicine, Molecular Biology, Immunology, Transfection, antibodies, neuron, immunocytochemistry, fluorescent microscopy, autoimmunity
A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting
Institutions: Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Liverpool School of Tropical Medicine, New York University School of Medicine.
causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum
-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum
has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1
. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2
and restrict their environment to a more favorable low oxygen pressure 3
. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro
, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5
. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6
. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8
has also been associated with severe disease 9
One of the most recently described P. falciparum
cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro
. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10
. Whether clumping occurs in vivo
remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11
. In addition, the ability of clinical isolate cultures to clump in vitro
was directly linked to the severity of disease in Malawian 12
and Mozambican patients 13
, (although not in Malian 14
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15
. Here, we present a method for in vitro
platelet-mediated clumping of P. falciparum
with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.
Infection, Issue 75, Infectious Diseases, Immunology, Medicine, Microbiology, Molecular Biology, Cellular Biology, Parasitology, Clumping, platelets, Plasmodium falciparum, CD36, malaria, malarial infections, parasites, red blood cells, plasma, limited resources, clinical techniques, assay
Preparation and Pathogen Inactivation of Double Dose Buffy Coat Platelet Products using the INTERCEPT Blood System
Institutions: Örebro University Hospital.
Blood centers are faced with many challenges including maximizing production yield from the blood product donations they receive as well as ensuring the highest possible level of safety for transfusion patients, including protection from transfusion transmitted diseases. This must be accomplished in a fiscally responsible manner which minimizes operating expenses including consumables, equipment, waste, and personnel costs, among others.
Several methods are available to produce platelet concentrates for transfusion. One of the most common is the buffy coat method in which a single therapeutic platelet unit (≥ 2.0 x1011
platelets per unit or per local regulations) is prepared by pooling the buffy coat layer from up to six whole blood donations. A procedure for producing "double dose" whole blood derived platelets has only recently been developed.
Presented here is a novel method for preparing double dose whole blood derived platelet concentrates from pools of 7 buffy coats and subsequently treating the double dose units with the INTERCEPT Blood System for pathogen inactivation. INTERCEPT was developed to inactivate viruses, bacteria, parasites, and contaminating donor white cells which may be present in donated blood. Pairing INTERCEPT with the double dose buffy coat method by utilizing the INTERCEPT Processing Set with Dual Storage Containers (the "DS set"), allows blood centers to treat each of their double dose units in a single pathogen inactivation processing set, thereby maximizing patient safety while minimizing costs. The double dose buffy coat method requires fewer buffy coats and reduces the use of consumables by up to 50% (e.g.
pooling sets, filter sets, platelet additive solution, and sterile connection wafers) compared to preparation and treatment of single dose buffy coat platelet units. Other cost savings include less waste, less equipment maintenance, lower power requirements, reduced personnel time, and lower collection cost compared to the apheresis technique.
Medicine, Issue 70, Immunology, Hematology, Infectious Disease, Pathology, pathogen inactivation, pathogen reduction, double-dose platelets, INTERCEPT Blood System, amotosalen, UVA, platelet, blood processing, buffy coat, IBS, transfusion
In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis
Institutions: University Hospital of Strasbourg, University of Strasbourg, University Hospital of Strasbourg.
Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are intensively and increasingly recruited in septic joints from the early phases of infection and the infiltration is supposed to regress once efficient removal of the pathogens is obtained. The ability to identify in vivo
macrophage activity in an infected joint can therefore provide two main applications: early detection of acute synovitis and monitoring of therapy.
noninvasive detection of macrophages can be performed with magnetic resonance imaging using iron nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO). After intravascular or intraarticular administration, USPIO are specifically phagocytized by activated macrophages, and, due to their magnetic properties, induce signal changes in tissues presenting macrophage infiltration. A quantitative evaluation of the infiltrate is feasible, as the area with signal loss (number of dark pixels) observed on gradient echo MR images after particles injection is correlated with the amount of iron within the tissue and therefore reflects the number of USPIO-loaded cells.
We present here a protocol to perform macrophage imaging using USPIO-enhanced MR imaging in an animal model of septic arthritis, allowing an initial and longitudinal in vivo
noninvasive evaluation of macrophages infiltration and an assessment of therapy action.
Medicine, Issue 80, Biomedical Engineering, Anatomy, Physiology, Molecular Biology, Diagnostic Imaging, Musculoskeletal System, Bacterial Infections and Mycoses, Macrophage, MR imaging, infection, arthritis, USPIO, imaging, clinical techniques
Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice
Institutions: University of Illinois at Chicago , University of Illinois at Chicago .
Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2
During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3
Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4
It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5
Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4
Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil β2 integrins and firm adhesion between two cell types. Despite the advances in in vitro
experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7
those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-α-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.
Immunology, Issue 74, Medicine, Cellular Biology, Molecular Biology, Inflammation, Hematology, Neutrophils, Microscopy, Video, Thrombosis, Platelet Activation, Platelet Aggregation, Intravital microscopy, platelet, neutrophil, rolling, adhesion, vascular inflammation, thrombus formation, mice, animal model
In Vivo Modeling of the Morbid Human Genome using Danio rerio
Institutions: Duke University Medical Center, Duke University, Duke University Medical Center.
Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo
complementation in zebrafish. Zebrafish (Danio rerio
) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo,
and can be genetically manipulated.1
These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.
Molecular Biology, Issue 78, Genetics, Biomedical Engineering, Medicine, Developmental Biology, Biochemistry, Anatomy, Physiology, Bioengineering, Genomics, Medical, zebrafish, in vivo, morpholino, human disease modeling, transcription, PCR, mRNA, DNA, Danio rerio, animal model
Christopher Hughes: An in vitro model for the Study of Angiogenesis (Interview)
Institutions: University of California, Irvine (UCI).
Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.
Cellular Biology, Issue 3, angiogenesis, fibrin, endothelial, HUVEC, umbilical, Translational Research

domain: medical


text:
Netflix announces upcoming comedy about a gay man living with cerebral palsy.
After producing LGBTQ-themed shows like Sense8, Sex Education and Grace and Frankie, Netflix landed a deal with Jim Parsons to make Special, a comedy about a gay man with cerebral palsy. Based on a memoir by Will & Grace writer Ryan O’Connell, the show will focus on life as a queer disabled person.
Based on O’Connell’s memoir I’m Special: And Other Lies We Tell Ourselves, the show is set to premiere on April 12. The semi-autobiographical comedy is about “a gay man with mild cerebral palsy who decides to rewrite his identity as an accident victim and finally go after the life he wants.”
O’Connell’s I’m Special: And Other Lies We Tell Ourselves was published by Simon & Schuster in 2015 to critical acclaim. The actor-writer-producer served as editor of Thought Catalog and has contributed to Vice, BuzzFeed and numerous other publications. His writing career spans television as well, having recently served as an executive story editor on NBC’s revival of Will & Grace. O’Connell also was on the writing staff of MTV’s Awkward.

domain: entertainment


Instagram is a popular photo sharing app with over 50 million users (probably more now), according to mashable.com. One thing these millions of users have in common is they either own an iPhone or Android. No, there was no survey taken to see what phone these users have. It’s because Instagram is a mobile platform and currently, you can only download the Instagram app if you have an iPhone or Android.
What if you have an Instagram account already and want to view Instagram photos on the web? Even though Instagram is meant to be used on your phone, there are a few ways to view Instagram photos on the web.
Here are 3 ways to view Instagram photos on the web:
Hootsuite.com is a social media dashboard to manage and measure your social media networks. You can manage social media platforms such as Twitter, Facebook fan pages and profiles, LinkedIn and many more.
Hootsuite has also added Instagram to its app directory. (Other apps include SlideShare, HubSpot and more.) With this Instagram app, you can view people you are following’s photos, like and comment on the photos. You can also view your own photos, popular photos, photos you have liked and use the search feature.
Webstagram.com is another website that allows you to view Instagram photos on the web. Very similar to the Hootsuite app’s functions, this website allows you to view people you are following’s photos, like or comment on photos and use the search feature.
Additional features include the ability to follow and unfollow users and see your followers and who you are following.
Statigram.com is a little different than the other websites. There are a lot of additional features.
Just like Hootsuite and Webstagram, Statigram allows you to like, comment, follow and unfollow, view popular photos… all the functions Webstagram has.
However, the additional features that I find really cool are the statistics. For example, they will create reports to show you how many likes or comments you have received, which photos are the most popular, what filters you have used the most, filters that get the most likes, which hashtags you used and much more!
When you give them your email address, they will send you monthly reports. For example, here is a photo they sent me about my “firsts” on Instagram:
They even sent an “IG Birth Certificate” (in other words, when I posted my first photo):
Before you use any of these websites, make sure you have created an Instagram account using an iPhone or an Android. The only negative side of these ways to view Instagram photos on the web is that you cannot post photos. You can only view, like and comment. Maybe that will change one day.
For more info about Instagram please visit this page.
Bethany Looi is a teen who helps Internet Marketers with their VA and social media client care needs. She loves social media, food photography, traveling and the Spanish language. Bethany is combining these passions to pursue a career in journalism and social media management. She has finished high school at the age of 16 and is currently taking a year off to pursue her passions and growing her business.
Photo credit: Instagram logo- http://instagram.com/


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Their love can drive the distance.
Isla Moody’s foster family had waited two years to be able to formally adopt her. But when the big day finally came, the coronavirus pandemic prevented them from celebrating in the way they’d planned.
Evan and Cayela Moody had barely had their foster-care license for a month in 2018 when they got a call that a premature, 4-pound baby needed a home, reports “Good Morning America.”
After picking up the newborn from the hospital, the idea of permanently parenting was quickly proposed.
“We’d had Isla for 48 hours and somebody asked if she needed us for adoption, would we want to?” Cayela tells the outlet. “I brought it up to Evan and he said, ‘You won’t need to ask me again. If she needs us for adoption, I’m all in.’ ”
In February, after years of uncertainty, the family excitedly learned the adoption had officially gone through. But COVID-19 was already rapidly sweeping the globe and the actual adoption ceremony was put indefinitely on hold, the family’s local Fox News outlet reports.
On April 30, the big day at last arrived: After 700 days in their foster care, Isla was formally adopted by Evan and Cayela in a Zoom ceremony. The parents and their four biological children attended from their Jacksonville, Florida, living room while some two dozen friends and family were also present, through the airwaves, for the moment Isla became a Moody.
Not satisfied with the virtual event, the Moodys decided to also hold a real-world happening while also being mindful of pandemic etiquette. With the help of foster-care support organization Fostering Hope, they threw little Isla a car parade.
“Everybody wanted to be part of Isla’s adoption, but I thought, ‘I can’t do this right now, we’ll throw a party later,’ ” says Cayela, “A friend asked if it would be OK to throw a parade, and I said that’d be fine. I wanted people who supported us and Isla to be able to celebrate because they’ve been part of this, too.”
Dozens of cars drove by the family’s home in celebration of Isla, who her parents describe as “easygoing” and “articulate,” with a “beautiful” personality.
“Whether or not she had understanding at all, or to what degree she understood what was going on, we’ll be able to look back at that day and say you were really loved by a lot of people,” says Evan. “And yeah, you were adopted during a pandemic and that was super weird, but that being said, you didn’t just have a party, you had a parade.”


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The McAdams Remodeling & Design Process
The start of the new year seems like a good time to revisit our Design Process for those new to the McAdams Remodeling & Design family. Our process is pretty simple, we want you to feel like your job is the only one we’re working on. A typical McAdams project will follow these steps:
- Initial Meeting and Evaluation. After our initial phone call, we will schedule a meeting at your home where we can better evaluate the proposed project and get to know each other better. It’s an opportunity for both of us to ask questions and allows us to physically see your home and discuss with you any potential issues along with the scope of work in greater detail.
- Feasibility Study. This step ensures we do our due diligence around what can be done to your home based on city building restrictions, zoning issues and more. Though it’s not necessary in every situation, more involved remodels benefit from rough architectural drawings and analysis of potential challenges.
- Letter of Intent. Once you’ve reached this step, it means we’ve mutually agreed to move forward, and the design process will begin. Project specifics get entered into our online project software, BuilderTrend, and your design plans come to life with the materials you select. This is typically when we submit for permits on your behalf.
- By the time we reach this stage, you’ve been given an estimated investment range, we’ve developed solid plans and permits have been requested. With all of that detailed information, we can develop a fixed-price contract which outlines expected costs of materials and labor and overall commitments on your project. You will receive an approximate start date, we’ll develop your project calendar, and materials with long lead times, like cabinets, will be ordered.
- Construction Begins. Every project begins with a pre-construction meeting. We’ll discuss expectations, answer any questions, and ensure we have the information necessary to lessen as many interruptions as possible to your daily life.
Our goal is to be your favored remodeler today and in the future. We aim to develop excellent relationships with our clients so that you can’t help but rave about us to your friends and family. We’re so committed to this idea that we offer a 10-year Limited Warranty. If you have more questions about what it’s like to work with us, don’t hesitate to reach out.
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