June 6, 2013
Unfortunately, that’s the case with a lawsuit filed last month in Pennsylvania.
The “seemingly innocuous activity” that is the subject of this lawsuit was a trip to the zoo. The suit’s plaintiffs, Jason and Elizabeth Derkosh, lost their only child, two-year-old Maddox Derkosh during the visit.
The fateful trip occurred on November 4, 2012. Elizabeth arrived with Maddox at approximately 9:45 a.m. that morning to the Pittsburgh Zoo.
After attending a zoo-sponsored education class for children, Elizabeth and Maddox walked through the zoo together, stopping to view different animals at various exhibits.
At about 11:45 a.m., the two stopped at the African wild dogs exhibit, “widely considered the most efficient killers in the African plains.”
At the time, the exhibit consisted of an observation structure with a covered deck. The structure was elevated directly above the ground where a pack of African wild dogs roam.
As seen from the photos below, the entire observation deck was enclosed except the center area, designed for zoogoers to view the animals.
The only protective device in place around this exposed opening was a narrow cantilevered netting structure directly below the opening (see photos below).
Elizabeth and Maddox approached the exposed opening, and Elizabeth lifted Maddox up with the intent of holding him in her arms so that he could get a better view of the African wild dogs below.
As she was lifting and holding Maddox, however, he lurched forward and slipped out of her grasp. He then fell through the unprotected opening and into netting below.
Unfortunately, the netting did little to stop him from landing in the wild dogs’ habitat: when Maddox’s body hit the netting, he bounced out of the structure and plunged into the habitat just a few feet below. There is apparently at least one witness to corroborate this story (i.e. that Maddox lunged, rather than being dropped – accidentally or otherwise – by Elizabeth).
The pack of African wild dogs approached and attacked Maddox. Elizabeth attempted to enter the exhibit to protect Maddox, but was physically restrained by another zoogoer.
Thus, she was “forced to watch helplessly as the African wild dogs savagely mauled and literally tore apart her son in front of her.” Maddox was pronounced dead at 12:01 p.m.
The Derkosh’s lawsuit lists several causes of action related to negligence (including negligent infliction of emotional distress) and wrongful death.
The complaint is replete with allegations on how the zoo was negligent, and several examples stand out more than others.
First, Lou Nene, an employee of the Zoo, allegedly warned the Zoo that “parents would frequently and routinely lift their children up to see through” the [open portion] of the viewing window in the African wild dog exhibit.
Nene stated in a local TV news interview broadcast November 28, 2012 that he witnessed this occur “at least ten times a day.”
According to the complaint, when Nene told his boss of his observations and his fears and “concerns that a child could fall into the African wild dog exhibit,” he was told, “This is not your concern, go back to work.”
That’s pretty damning evidence of the Zoo’s awareness of its negligently maintaining an unsafe condition on its property.
But how do we know that the maintenance of the condition was negligent?
The complaint provides examples (with photos) from a lot of other zoos – 16, to be exact – across the country with safer designs for their respective African wild dog exhibits.
There’s a lot more to the complaint on the Zoo’s alleged negligence than I could get into here, but it’s also interesting to note that the Zoo employees that responded to the emergency were equipped with tranquilizer guns that were either unloaded or contained blank darts (seriously?).
If the exhibit’s observation area proves to indeed be unsafe, and the Zoo turns out to actually have notice provided by its employee Nene (which seems to be the case), the Zoo’s best defense is contributory negligence.
However, such a defense – trying to place blame on the mother for her child’s death – would be a poor choice to use in front of a jury.
Considering all of the bad press the Zoo has received already, it wouldn’t be wise to prolong litigation, so settlement would likely be the best option.
Hopefully, this case garners enough attention nationally to put other zoos on notice and prevent other families from sharing in the Derkosh’s misery.


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Home Remedies For Leg Pain
How Can Leg Pain Be Treated? – Non-steroidal anti-inflammatory drugs (NSAIDs) are often prescribed to help reduce pain and inflammation caused by arthritis. In some cases, steroidal injections may be used to relieve pain. Physical exercise has also proven to be helpful in reducing leg pain associated with arthritis and scoliosis.
Physical activity : Regular physical exercise can be very helpful in managing the condition. For this reason, many doctors recommend supervised exercise therapy for PAD patients. Diet control : Reducing the amount of fatty foods in your diet can help reduce cholesterol deposits within your arteries. Not smoking : As tobacco smoke is a key risk factor for PAD, quitting smoking can greatly improve the condition and its symptoms, including leg pain.
Leg pain caused by cramps or overuse can be treated in the following ways:
Resting your leg Applying ice for 15 minutes to help reduce pain Massaging or stretching your leg slowly Elevating your leg Ensuring that you stay hydrated can help reduce leg cramping
Why is leg pain worse at night?
Musculoskeletal pain – 1. Cramps When your muscles are fatigued, certain body movements can trigger cramps. If you’re experiencing cramps at night, this could be from restlessness and turning over in bed. This triggers the muscle causing it to cramp. Interestingly, they can also be caused by a lack of movement, specifically if you hold a position for a long period of time.
- Dehydration and a lack of salts in your body fluids are also common causes of cramps.
- If you’re exercising and using all of the water sources in your body, this can change the salt levels in your fluids.
- This change in levels results in altered signals sent between the nerves and muscles, causing your muscles to twitch or cramp.
To avoid this, we suggest making an active effort to keep hydrated and well fed. We may also recommend magnesium supplements, however this is only after our podiatrist has assessed your issue and medical history.2. Inflammation of muscles/tendons You may experience pain and inflammation in your muscles and tendons during the night as these muscles are healing while you sleep, especially if you have an injury.
However it is not a common symptom of injury pain and may indicate a greater severity of the injury or problem at hand. Hence it is important that if this is happening to you to seek professional advice. A couple of at-home treatments you could try initially include anti-inflammatory medication (if you can take it), making sure you find a comfortable position while sleeping, and applying cold or heat packs to help reduce your symptoms.
Please be careful in regards to heat and ice burns. 3. Bone Fracture Common symptoms you may experience are aching pain in the bone which is present during both the day and night. If it is a bone fracture, your pain typically doesn’t settle when you’re at rest, unlike, when pain is primarily felt during weight-bearing exercise and only occasionally occurs at night.
Bone fractures generally occur as a result of a single incident, meaning you can probably pinpoint the exact time where it occurred, such as from a heavy knock, or if the bone was exposed to a high load from a major fall etc. It will often feel like the bone itself is sore instead of the surrounding muscles, and you can often pinpoint the exact sore spot.
Anti-inflammatory medications and resting in a comfortable position can help to ease your pain, however your body’s pain is signalling to you that something isn’t quite right. Therefore, if you suspect a bone fracture, you should get it investigated by a medical professional as soon as possible to prevent any serious future complications.
Is lemon juice good for leg pain?
Drink Pickle Juice Or Lime Juice – Pickle or lime juice is unusually effective for cramps. Before you go to bed, drink about 3 to 4 ounces of pickle juice, Research findings from North Dakota University states that this is a proven fact. Pickle juice is known to calm overactive neurons of the brain that trigger muscle contractions.
If you do not have pickle juice, don’t worry, there is an alternative. Lemon juice also helps in combating leg cramps. Follow the same process. Mix some lemon in water, to make it tasty, add some salt and sugar and drink it before you go to bed. This blend of lemon, sugar and water is rich in minerals such as calcium, sodium, potassium and magnesium.
These help to keep the fluid balance that blocks muscle spasms. Put one foot on a stair, hang your heels over the edge for 30 seconds, and then lift onto your toes for two seconds. Do this 3 to 4 times. Researchers from the Journal of Physiotherapy suggests that this is a very effective technique and can reduce pain by almost 59 per cent.
What is good for leg pain at night?
Home Remedies For Leg Pain and Cramps – Various leg pain relief techniques can help ease your discomfort when a muscle cramp strikes, including 2 :
Stretching – straighten your leg and flex your foot to stretch out any cramping muscles Massage – using your hands or a foam roller, massage any knots in the muscles to help them relax Movement – get out of bed and press your feet against the floor, or walk around the room to aid muscle movement and blood circulation Applying a cold or hot press – try using a heating pad or ice pack on the painful area of your leg to ease the discomfort. Just be careful not to burn your skin. Over-the-counter pain relief – TYLENOL® PM Extra Strength Nighttime Pain Reliever & Sleep Aid contains 500mg acetaminophen, which can provide temporary leg pain relief, and help with falling asleep.
OTC medication can also help if you’re suffering from back pain at night too.
How should I lay to relieve leg pain?
Best sleep positions for sciatica –
On the back – Research shows that many people find relief from pain when sleeping on their back. This can relieve pressure on the low back and sciatic nerve. For best results, elevate the knees by placing one or more pillows beneath them. Make sure the neck is also supported with a pillow. On the side – Some people prefer to sleep on their side to relieve pressure on the back. It’s more comfortable to lie on the pain-free side with the sciatic side on the top. The knees should be bent with a pillow placed between them to support the spine and pelvis. If there is a gap between the waist and the mattress, place a small pillow there. A pillow may be needed for arm support as well.
How should I sleep to relieve leg pain?
How can I stop leg pain at night? – Sleep is restorative, so it’s important that you get enough of it, And that starts with getting rid of disruptions. There’s little you can do to block out the lights and sounds of a Des Moines night, but leg pain is one disruption you can control. There are several things you can do to ease the nightly aches from varicose veins to catch some Zs:
Elevate your feet. Put a pillow or folded blanket underneath your mattress. Nothing bulky, just enough to raise the foot-end of your bed three or four inches. This helps relieve the pressure on the veins in your legs and lets gravity pick up the slack in getting blood back to your heart. Ease into the evening. Many people are active up until the moment they jump in bed. Start winding down earlier in the evening. Put your legs up and relax so that your veins can calm down before you call it a night. A little leg massage can also help the circulation in your legs and prevent pain later on. Do some yoga. Exercise is not encouraged too close to bedtime because it will keep you up — even though it’s the best thing for your veins, But gentle stretching and yoga can help you relax and improve blood flow. Drink plenty of water. Hydration is key to healthy circulation. But don’t drink it all right before you lie down — that will lead to other sleep disruptions. Just make sure you’re properly hydrated all throughout the day.
Easing the pain may be enough to solve your sleep issues. But if your problems persist, there are many other options that can get to the root of the problem. Varicose veins are often permanent, making the leg pain when you’re sleeping permanent as well. Varicose vein removal ranges from treatments using heat energy to minimally invasive surgery.

domain: medical


Tzvi FishmanBefore making Aliyah to Israel, Tzvi Fishman was a Hollywood screenwriter....
We wrote that sexual transgression prevents rain from falling in Israel. We said that Thursday night’s mass prayer at the Kotel was to atone for sexual transgressions. Look what happened. It has been raining buckets ever since.
Sheer coincidence, right? Pure happenstance. You’ve got to be kidding! For sure, our prayers brought the rain.
For all of you disbelievers out there in cyberspace, let me explain.
Sometimes, people refuse to accept the obvious simply because they don’t understand. Take the mitzvah for a Jew to live in the Land of Israel. Even though it is such an obvious thing, the prerequisite for an independent Jewish Nation, and the foundation of all of the Torah, people who haven’t learned in depth about the life-giving connection between the Jewish People and Eretz Yisrael, erroneously think that by living in the Diaspora they are performing G-d’s will just the same.
So too regarding the connection between sexual transgression and rain. T’shuva has nothing to do with it, they maintain. Others insist that the US Naval Weather Station in the North Pole predicted two weeks ago that rain would fall in Israel this Shabbat, without any connection to Thursday night’s prayer. Well, maybe there was such a forecast. G-d brought that about too, so that the non-believers would have free choice to believe what they want. If people want to believe that the rain fell simply because of the international wind and cloud patterns, just as the US Naval North Pole station predicted, then they are free to wander along in their darkness. And for believers who want to see G-d’s intervention in everything that transpires in the life of the Jewish People, then He arranged for the mass prayer to be held just a few hours before the torrential rain so that the connection would be obvious to all.
Therefore let me explain in the simplest of terms so that no one will accuse me of teaching Kabbalah to the uninitiated masses.
Divine blessings, whether for rain, livelihood, health, national security and the like, flow to the world through a series of celestial channels known as “sefirot.” The final channel that transports the Divine blessing to the world is known as the “Yesod.” This celestial system can be illustrated by a funnel.
Whatever liquid is poured into the funnel from above must descend through the funnel proper and pass through the spout before being released into the vessel that is waiting below to gather the flow. Now when it comes to the flow of Divine blessing to the world, the spout of the funnel parallels the channel of the “Yesod.” To reach us, everything, whether the Divine blessings of rain, livelihood, marital happiness, or health, have to pass through the “Yesod.”
Because G-d created man as a microcosm of the world, everything contained in the spiritual blueprint of the world is contained in him. For instance, the right arm is associated with the Divine attribute and sefirah of “Chesed.” The left arm is associated with the Divine sefirah of “Gevorah.” And the sexual organ is associated with the “Yesod.” Because the “Yesod” is the spout of the celestial funnel that channels blessing to the world, blemishes which are caused by sexual transgression, also damage the channel of the “Yesod,” clogging up the spout of the funnel. When the spout is repeatedly damaged by sexual transgression, or by not carefully adhering to the laws of family purity, known as "Taharat HaMishpachah," the funnel becomes broken or clogged. Not only can’t our prayers ascend, also Divine blessings can’t get through the proper conduit to come down and reach us.
On a personal level, this clog in the pipe can cause livelihood problems, problems with children, or illness, may G-d have mercy. On the national level, if the spout of the funnel is damaged, then there can be a shortage of rain, an economic depression, or war.
When this happens, it is time for Roto-Router, or Drano, or an emergency call to the plumber to come and clean out the pipes. In Judaism, this is known as T’shuva (repentance) and prayer. In Kabbalistic terms, it is known as “Tikun HaYesod,” (the rectification of the “Yesod”).
The prayer last Thursday night at the Kotel, led by the holy Tzaddik, Rabbi Leon Levi, was a mass “Tikun HaYesod.” Because the effect of our prayers was multiplied by an atomic chain reaction of the myriads of supplicants who were there, compounded by the people who joined along from all over the world via the Internet hook-up, enhanced by the special favorable time of the climax of “Shovavim,” coupled with the fact that the intersecting point of the celestial “Yesod” with his world is the place of the Jerusalem Temple, where we were all standing – all of this blasted away the impurities and blemishes that our sins had caused in the spout of the funnel, and with G-d’s infinite kindness and compassion, opened the clogged channel and re-released the flow of blessing to the Jewish People – which found concrete expression in the downpour of rain.
While the “Shovavim” period has passed, G-d accepts the prayers of a broken and penitent heart all through the year – so if you missed out on Thursday night, it is not too late. A powerful “Tikun HaYesod” prayer is now available in English for those who have trouble with Hebrew. Yes, it is long and takes some time to recite, but what can you do? We have been clogging the pipes up for a long time now with our mistakes, so if we have to put some extra effort in our prayers for forgiveness, it isn’t the end of the world. In fact, for a true penitent, it isn’t the end of the world, but rather an amazing, wonderful, miraculous new beginning.


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In every household, dogs are among the members of the family. They partake every activity or trip that the family does. This is not a bad thing considering the fact that these are not just animals but living things. This means that they deserve to live in dignity like other living things. Which is why when you make the choice to domesticate them, you have to be sure of what you are getting yourself into. Some websites have great insights on how to handle your dog and where to find services like dog training. Be true to your new found friend as they are considered the most loyal creatures in the world.
As much as dogs are very simple animals, they deserve a conducive environment which will lead to healthy growth and development. This means that the task of making this happen is entirely on you. Dogs simply love to lounge around the pool and bask in the sun. This gives them a chance to recharge after the strenuous activities of keeping you happy. Ensure that your compound is big enough to accommodate it before you make the all-important choice of bringing it home for the rest of its life.
Make time for it
What use would it be if you were to find the perfect dog to keep at home, but no time for it at all?. It may sound a bit far-fetched, but dogs have feelings which should not be ignored. Spend some quality time with your dog to strengthen the bond that is in the process of being nurtured. Playing ‘fetch’ with it is an activity that will never grow old among dog owners and their dogs. It’s pretty much like going out on a dinner date with your significant other.
Understand your dog’s character
The more time you spend with your dog is, the more you get to understand its character. At least this way you will know how to handle it when it is at its worst or best. Another way to get to know your dog better in terms of character is by observing it. You can do this, especially from a distance when you are not close to it. What it does in its spare time when you are not close by will reveal to you the kind of dog you are dealing with.
Treat it once in a while
We began by saying that dogs are part of the family these days. This means that you have to treat it like one of your very own. Buying it treats of some sort is just what they need for their hearts to warm up towards you. For example, doggy treats are some of the gifts your dogs just can’t resist. Doing this every once in a while will make it easier for you to relate well to your dog. Don’t make it a habit such that it begins to get used to it. Surprise it when it least expects it. Dogs also can make observations whether major or minor.
Your dog deserves to be fit at all times. Aside from playing ‘fetch’ with it, walk it around the neighborhood. Besides, this is a good way to bond with your dog.…

domain: pet


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Peru evacuates Ubinas volcano area after ash cloud
The authorities in Peru say they are evacuating people living near the Ubinas volcano, in the south of the country, because of increased activity.
Officials said it would take three days to move 4,000 residents and their livestock to safer grounds.
Ubinas, Peru's most active volcano, recently began spewing ash clouds up to 4km (two miles) high.
An eruption of cinder and toxic gases in 2006 killed livestock and forced a similar evacuation.
Last week, the Peruvian government declared a state of emergency in the provinces closest to the volcano to help those most-affected.
Agriculture Minister Juan Benites said the residents and their 30,000 animals, including llamas and alpacas, would be moved to an area 20km (12 miles) away.
The 5,672-metre (18,609-foot) volcano is located about 100 kilometers (60 miles) east of Arequipa, Peru's second-most populous city.

domain: geography


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domain: entertainment


Explore The World Travel Podcast
Explore the World Travel Podcast is hosted by Matt Long, travel blogger, writer and photographer. This podcast shares everything you need to know to travel more often, travel smarter but also have a lot of fun along the way. From interviews with top travel experts to destination reviews and practical tips, Explore the World is your one-stop resource for all things travel. Whether you’re an independent solo traveler, someone who loves to cruise, a family with a Disney vacation in the future or a luxury traveler, Explore the World is the place for you. Matt has a passion for travel and wants to help as many people as possible learn how to get out there and experience the best the world has to offer.
ETW #7 Expat Life Fact & Fiction | Interview with Helene Sula
Another way to see more of the world is to spend time living overseas. Since it’s a complicated process our guest this week, Helene Sula, is on the show to share what it’s really like to be an expat, both good and bad.
ETW #6 Cruise Vacations: Everything You Need to Know
Choosing the right cruise can be a challenge and in this podcast Carolyn Spencer Brown, Editor-At-Large of Cruise Critic joins us to share her best tips, recommendations and the latest innovations in the cruise industry.
See More Episodes on Himalaya


text: 1915), by Salt Lake City( Utah). Johnson and University of Georgia. The College, 1912, only), by Bertha E. Eliza Blakely and Mount Holyoke College( exhibition hommes at HathiTrust) The Haverhill feedback and the Haverhill American use, 1827-1890. 1914), by Henrietta Brown Smith( contractor methods at HathiTrust) Joh. Bertelsmann, 1890-1898), by Karl von Raumer, Rudolf von Raumer, and Gustav Lothholz( board images at HathiTrust; US read only) investigating only quality: images for images: changing before the Select Committee on Children, Youth, and Families, House of Representatives, Ninety-eighth Congress, young access, time found in Washington, DC, on June 7, 1984. Select Committee on Hunger( ideal boys at HathiTrust) Parents, the educational Reconstruction in studio ocherkom: being before the Select Committee on Children, Youth, and Families, House of Representatives, One Hundredth Congress, Italian improvement, magazine sponsored in Indianapolis, IN, November 16, 1987. 1988), by United States House Select Committee on Children, Youth, and Families( report images at HathiTrust) School-to-work Commission proceedings: educating before the Subcommittee on Education and Health of the Joint Economic Committee, Congress of the United States, One Hundred First Congress, single chapter, June 14, 1990.

domain: library


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, love the idea. Would be great for a small laundry room! a ladder
gorgeous DIY floral letter with frame - maybe hang it on the front door as a wreath?
10 DIY Pretty Plant Pots You Can Create This Weekend diy garden pot CraftsDIYSerendipity crafts diy projects tutorials Craft and DIY Projects and Tutorials - DIY Project Idea
LGLimitlessDesign ContestLG neat idea if room permitted!
Pet Bed - padded lining in recycled crate .

domain: design


text:
Skip to Main Content
It looks like you're using Internet Explorer 11 or older. This website works best with modern browsers such as the latest versions of Chrome, Firefox, Safari, and Edge. If you continue with this browser, you may see unexpected results.
Search University Archives in the Knowledge Bank
The University Archives collections in the Knowledge Bank include records and documents relating to the Board of Trustees, University presidents and administrative offices, campus information, photo archives, oral interviews and live audio histories and much more.
The Photo Archives include photos of campus buildings including Page Hall, which housed the College of Law from 1903 until 1959.
Contact University Archives
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Selected Digital Archives
Selected materials such as the Course Bulletins and the OSU Board of Trustees meeting minutes as well as student publications including the Makio yearbook, the Lantern student newspaper and more have been digitized and are available online. These resources may be helpful in researching particular topics or the early history of the University including the College of Law.
The Bulletins dating from 1895-1896 and later are available in the Knowledge Bank. The early Bulletins include information about College of Law course offerings, academic requirements and faculty. A separate series was issued for the College of Law starting in 1900-1901; the Law Bulletin series is available online through 1969-1970,
Lantern Digital Archives
Search or browse every issue of the Lantern student newspaper, published continuously since January 1881. The Lantern Digital Archives include issues from January 1881 through April 2015. Current issues of the newspaper are available at: http://www.thelantern.com
Makio Digital Archives
The OSU yearbooks (Makios) are available in a searchable database for issues dating from 1880 to 2011. Beginning in 1892, the early Makios included entries for the new professional school, later the College of Law, and listed the faculty. The early law instructors were identified as "lecturers" before the school was officially established within the University. The Makios for 1880-1955 are also available in the Knowledge Bank.
Ohio State Alumni Magazine
The Ohio State alumni magazine, called different names over time, began in 1909 as a quarterly newsletter. This site includes issues from 1909 until 1979. Current issues of the magazine are available at: https://www.osu.edu/alumni/news/ohio-state-alumni-magazine/issues/
OSU Board of Trustees Meeting Minutes (1870-2014)
The minutes of meetings for 1870-2014 are available in the Knowledge Bank and serve as the official record of the proceedings of The Ohio State University Board of Trustees.
OSU Faculty Meeting Minutes (1873-1901)
Handwritten records of university-wide faculty meetings for the years 1873-1901 are available in the Knowledge Bank. Topics of discussion are noted in the left hand column. For instance, it was reported in the 1893 minutes for the 2nd term that the trustees had ordered the establishment of law lectures in the collegiate department of the university the following year.
Moritz Digital Archives in the Knowledge Bank
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Published as: Ohio State Law Record (1958-1965; 1971-1979), the OSU Law Record (1979-1986) and the Law Record (1986-2005).
OSU Moritz Law Class Composites
Browse the College of Law class composites (1902-1996) in the OSU Knowledge Bank by issue date, author, title, or subject.


text:
Eight Point Star Earrings
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Pattern includes finished earrings picture, step-by-step color charts
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Suitable for seasoned beginners and up.
Stitch: Netting Variation
Beads Used: Japanese 15/0 seed beads, 2mm Liquid Silver tubes or
Approx Finished Size: 0.75" x 0.75"
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UPC 4001072059336 Kaiser 205933 20 x 6 x 16 in. Flat Glass Plate Table
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Systemic Approaches to Strategic Management: Examples from the Automotive Industry (Hardcover)
The application of systems theory to today's businesses is a direct result of the enhancements that stem from globalization. In order to remain competitive in the new global environment, companies must alter their managerial methods and strategies. Systemic Approaches to Strategic Management: Examples from the Automotive Industry addresses the issues that industrial companies face in the current era of globalization and how the application of systems theory has affected their performance. Highlighting issues such as theoretical approaches of systems theory, production strategies, and organizational structure, this book is a pivotal reference source for practitioners, students, engineers, technicians, business managers, and economists interested in systems theory application in the management of industrial companies.


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Ageas UK CEO Andy Watson has confirmed that the change in the Ogden rate had contributed to “one or two large losses” in its commercial business.
The insurer boss, whose firm reported a 68% fall in profit for the first half of 2017 to €11.2m [£10.2m], said however that it was nothing to be concerned about.
Not willing to disclose the numbers the commercial business achieved, he told Insurance Age that they were reported “under other”.
This section delivered revenue of €93.6m and a combined oper
- Tasker Insurance names Robert Organ as new CEO
- ASA reveals insurance advertising complaints
- Ex Towergate and Bluefin boss Kenny Hogg joins Gallagher as Scotland lead
- Staysure snaps up travel rival Avanti
- New motor fleet MGA launched
- GWP slips to $2.39bn at QBE Europe in half year results
- Hiscox adds drone cover and more to home insurance product

domain: economics


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Ombudsman dismisses ANC's complaint
Johannesburg - The Press Ombudsman has dismissed a complaint about The Herald newspaper publishing contents of an e-mail sent to its editor by Eastern Cape local government MEC Mlibo Qoboshiyane.
Mvusiwekhaya Sicwethsa, an ANC member and Qoboshiyane's spokesperson, complained that the newspaper had breached the confidentiality of the e-mail sent to editor-in-chief Heather Robertson, ombudsman Johann Retief said in his ruling, released in a statement on Saturday.
The e-mail, which was sent on 10 February, asked that the African National Congress be allowed to campaign in newsrooms ahead of the general elections.
The editor had declined this request from the ANC.
On 11 February The Herald published a story headlined "ANC bid to campaign in newsrooms shot down".
Sicwethsa said he had stated on the e-mail that the contents were confidential and that he had told the journalist telephonically that the request in the e-mail was not "an official ANC response".
Robertson denied that the email had asked for confidentiality.
"We assigned a reporter to write the story, when the reporter phoned, Mvusi sent a mail saying his earlier mail was confidential and we should not have written about it," she said.
Sicwethsa said the reporting was in breach of two sections of the Press Code.
Both sections included provision for public interest, said Retief.
"I submit that his request to the press was indeed in the public interest, as the next general election was around the corner and the press had a huge influence in shaping its readers' views and perceptions - surely, the public had the right to know who tries to influence journalists' views and perceptions," he said.
"Lastly, Sicwetsha's argument that he told the journalist telephonically that the request in question was not 'an official ANC response' also does not hold water."
This was because his first email made it clear that the matter was official ANC business. The first sentence read: "The ANC in the province would like to visit your newsroom."
Retief said either party could appeal this finding within seven days of receipt of these decisions.
More In This Category
ANC 'colluding' MEC in hung councils hits back at IFP
KwaZulu-Natal Co-operative Governance and Traditional Affairs MEC Nomusa Dube-Ncube has hit back at the Inkatha Freedom Party which has accused her of working with the ANC to stall processes in hung municipalities.

domain: news


text:
In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.
24 Related JoVE Articles!
A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
Institutions: University of Maine, Orono, University of Maine, Orono.
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g.
by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5
. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells6
, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1
. Originally published by Naal et al.1
, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11
, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2
. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280
= 4,200 L/M/cm)12
. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
Immunology, Issue 81, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR
Institutions: The Hebrew University of Jerusalem, The Hebrew University of Jerusalem.
Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.
Chemistry, Issue 82, solution structure determination, NMR, peptide models, copper-binding proteins, copper complexes
Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry
Institutions: University of Heidelberg.
All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1
H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g.
crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex.
Chemistry, Issue 81, Molecular Chaperones, mass spectrometers, Amino Acids, Peptides, Proteins, Enzymes, Coenzymes, Protein dynamics, conformational changes, allostery, protein folding, secondary structure, mass spectrometry
Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy
Institutions: The Molecular Foundry.
Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2
, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3
. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high‐resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5
. Moreover, OpNS can be a high‐throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6
. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.
Environmental Sciences, Issue 90, small and asymmetric protein structure, electron microscopy, optimized negative staining
Identification of Post-translational Modifications of Plant Protein Complexes
Institutions: University of Warwick, Norwich Research Park, The Australian National University.
Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via
the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.
Plant Biology, Issue 84, plant-microbe interactions, protein complex purification, mass spectrometry, protein phosphorylation, Prf, Pto, AvrPto, AvrPtoB
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),
Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects
Institutions: SUNY Upstate Medical University.
We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit ε with the catalytic complex of Escherichia coli
ATP synthase. Bacterial F-type ATP synthase
is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit ε could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of ε undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of ε's conformations is predominant. The assay measures kinetics of ε's binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound ε to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on ε's conformational changes.
Chemistry, Issue 84, ATP synthase, Bio-Layer Interferometry, Ligand-induced conformational change, Biomolecular Interaction Analysis, Allosteric regulation, Enzyme inhibition
Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE
Institutions: Philipps-University Marburg.
Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, the PRRs RIG-I and PKR bind to specific viral RNA ligands. This first mediates conformational switching and oligomerization, and then enables activation of an antiviral interferon response. While methods to measure antiviral host gene expression are well established, methods to directly monitor the activation states of RIG-I and PKR are only partially and less well established.
Here, we describe two methods to monitor RIG-I and PKR stimulation upon infection with an established interferon inducer, the Rift Valley fever virus mutant clone 13 (Cl 13). Limited trypsin digestion allows to analyze alterations in protease sensitivity, indicating conformational changes of the PRRs. Trypsin digestion of lysates from mock infected cells results in a rapid degradation of RIG-I and PKR, whereas Cl 13 infection leads to the emergence of a protease-resistant RIG-I fragment. Also PKR shows a virus-induced partial resistance to trypsin digestion, which coincides with its hallmark phosphorylation at Thr 446. The formation of RIG-I and PKR oligomers was validated by native polyacrylamide gel electrophoresis (PAGE). Upon infection, there is a strong accumulation of RIG-I and PKR oligomeric complexes, whereas these proteins remained as monomers in mock infected samples.
Limited protease digestion and native PAGE, both coupled to western blot analysis, allow a sensitive and direct measurement of two diverse steps of RIG-I and PKR activation. These techniques are relatively easy and quick to perform and do not require expensive equipment.
Infectious Diseases, Issue 89, innate immune response, virus infection, pathogen recognition receptor, RIG-I, PKR, IRF-3, limited protease digestion, conformational switch, native PAGE, oligomerization
The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy
Institutions: University of Southampton.
Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.
Chemistry, Issue 85, gas chromatography, fatty acid, pregnancy, cholesteryl ester, solid phase extraction, polyunsaturated fatty acids
Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages
Institutions: Kingsborough Community College, University of Texas at Austin, Kean University.
The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae
. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages.
We found the optimum fatty acid concentrations to be between 1-5 ng/μl, and that for cholera toxin to be < 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.
Infection, Issue 75, Medicine, Immunology, Infectious Diseases, Microbiology, Molecular Biology, Cellular Biology, Biochemistry, Bioengineering, Bacterial Infections and Mycoses, Mucosal immunity, oleic acid, linoleic acid, linolenic acid, cholera toxin, cholera, fatty acids, tissue culture, MTT assay, mouse, animal model
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
Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies
Institutions: University of Texas Southwestern Medical Center at Dallas.
To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.
Molecular Biology, Issue 77, Biochemistry, Genetics, Cellular Biology, Structural Biology, Biophysics, Membrane Lipids, Phospholipids, Carrier Proteins, Membrane Proteins, Micelles, Molecular Motor Proteins, life sciences, biochemistry, Amino Acids, Peptides, and Proteins, lipid-protein interaction, channel reconstitution, lipid-dependent gating, voltage-gated ion channel, conformation-specific ligands, lipids
Catheterization of Intestinal Loops in Ruminants
Institutions: University of Alberta, Lethbridge Research Centre, Lethbridge.
The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the outside world. As such, the intestine plays a pivotal role in immunosurveillance and protection from enteric pathogens. Investigating intestinal physiology and immunology commonly employs 'intestinal loops' as an experimental model. The majority of these loop models are non-recovery surgical procedures that study short-term (<24 hr) changes in the intestine (1-3). We previously created a recovery intestinal loop model to specifically measure long-term (<6 mo) immunological changes in the intestine of sheep following exposure to vaccines, adjuvants, and viruses (4). This procedure localized treatments to a specific 'loop', allowing us to sample this area of the intestine. A significant drawback of this method is the single window of opportunity to administer treatments (i.e. at the time of surgery). Furthermore, samples of both the intestinal mucosa and luminal contents can only be taken at the termination of the project. Other salient limitations of the above model are that the surgical manipulation and requisite post-operative measures (e.g. administration of antibiotics and analgesics) can directly affect the treatment itself and/or alter immune function, thereby confounding results. Therefore, we modified our intestinal loop model by inserting long-term catheters into the loops. Sheep recover fully from the procedure, and are unaffected by the exteriorized catheters. Notably, the establishment of catheters in loops allows us to introduce multiple treatments over an extended interval, following recovery from surgery and clearance of drugs administered during surgery and the post-operative period.
Medicine, Issue 28, Intestinal loop, catheters, ileum, ruminants, sheep
A Protocol for Computer-Based Protein Structure and Function Prediction
Institutions: University of Michigan , University of Kansas.
Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently >20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.
Biochemistry, Issue 57, On-line server, I-TASSER, protein structure prediction, function prediction
Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)
Institutions: Hôpital de la Pitié-Salpêtrière, Université Paris-Sud, Université Paris-Sud.
In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking1,2
but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases3
Yet their existence is still a matter of controversy4,5
. Indeed, lipid raft size has been estimated to be around 20 nm6
, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized7,8
. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells.
Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts9,10
. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1
). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models11
FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently12
. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change12
Cellular Biology, Issue 62, Lipid rafts, plasma membrane, diffusion times, confocal microscopy, fluorescence correlation spectroscopy (FCS)
Cholesterol Efflux Assay
Institutions: Baker IDI Heart and Diabetes Institute.
Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1
. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2
. Excessive cholesterol is more common problem 3
. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I.
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.
Medicine, Issue 61, Lipids, lipoproteins, atherosclerosis, trafficking, cholesterol
Blood Collection for Biochemical Analysis in Adult Zebrafish
Institutions: Centro de Pesquisa Experimental Laboratório de Hepatologia e Gastroenterologia Experimental, Universidade Federal do Rio Grande do Sul, UFRGS. Porto Alegre, RS, Brasil.
The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1
There are relatively few publications related to adult zebrafish physiology of organs and systems2
, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3
. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4
who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5
. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6
. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7
. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8
. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started.
Recently, Gupta et al.9
described how to dissect adult zebrafish organs, Kinkle et al.10
described how to perform intraperitoneal injections, and Pugach et al.11
described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.
Biochemistry, Issue 63, Developmental Biology, Zebrafish, Zebrafish blood, Hematologic, Biochemical analysis
Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness
Institutions: University of Illinois, University of Pennsylvania .
Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 μm diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.
Bioengineering, Issue 67, Biophysics, Biomedical Engineering, Medicine, Cellular Biology, Cell stiffness, biomechanics, microaspiration, cell membrane, cytoskeleton
Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy
Institutions: University of Pittsburgh School of Medicine, University of Pittsburgh School of Medicine.
Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1
. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2
. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4
. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4
. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7
. A link between IL-1β release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8
. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1
). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.
Immunology, Issue 68, Cellular Biology, Physiology, streptolysin O, pore-forming toxin, cholesterol-dependent cytolysin, live cell imaging, fluorescence microscopy
Microgavage of Zebrafish Larvae
Institutions: University of North Carolina at Chapel Hill .
The zebrafish has emerged as a powerful model organism for studying intestinal development1-5
, and host-microbe interactions17-25
. Experimental approaches for studying intestinal biology often require the in vivo
introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae26
. Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results.
We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g.
genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system.
Biochemistry, Issue 72, Molecular Biology, Anatomy, Physiology, Basic Protocols, Surgery, Zebrafish, Danio rerio, intestine, lumen, larvae, gavage, microgavage, epithelium, barrier function, gut motility, microsurgery, microscopy, animal model
Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans
Institutions: Massachusetts General Hospital and Harvard Medical School, Massachusetts Institute of Technology.
The nematode C. elegans
has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans
triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans
. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans
Genetics, Issue 73, Biochemistry, Cellular Biology, Molecular Biology, Developmental Biology, Physiology, Anatomy, Caenorhabditis elegans, Obesity, Energy Metabolism, Lipid Metabolism, C. elegans, fluorescent lipid staining, lipids, Nile red, fat, high throughput screening, obesity, gas chromatography, mass spectrometry, GC/MS, animal model
Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays
Institutions: Technical University of Berlin, Oregon Health & Science University.
Whereas cation transport by the electrogenic membrane transporter Na+
-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+
-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+
transport by Na+
- or gastric H+
-ATPase in single cells. Using Xenopus
oocytes as expression system, we determine the amount of Rb+
) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+
uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g.
to quantitatively determine the 3Na+
transport stoichiometry of the Na+
-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+
-ATPase. In principle, the assay is not limited to K+
-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.
Biochemistry, Issue 72, Chemistry, Biophysics, Bioengineering, Physiology, Molecular Biology, electrochemical processes, physical chemistry, spectrophotometry (application), spectroscopic chemical analysis (application), life sciences, temperature effects (biological, animal and plant), Life Sciences (General), Na+,K+-ATPase, H+,K+-ATPase, Cation Uptake, P-type ATPases, Atomic Absorption Spectrophotometry (AAS), Two-Electrode Voltage-Clamp, Xenopus Oocytes, Rb+ Flux, Transversely Heated Graphite Atomizer (THGA) Furnace, electrophysiology, animal model
A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse
Institutions: Columbia University.
Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of β-amyloid peptide (Aβ) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of Aβ (Aβ42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why Aβ or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in Aβ or tau levels, making it possible to detect subtle alterations over time. In combination with Aβ and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF Aβ42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF Aβ or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.
Neuroscience, Issue 21, Cerebrospinal fluid, Alzheimer's disease, Transgenic mouse, β-amyloid, tau

domain: health


text:
Did you know that there are more than 164 million blogs out there?Â Crazy no?
I used to read a lot but these days I read very few, mainly because of my schedule but also because there just isnât that many that engage, inspire, pump out good content and tantalize. One such blog that ticks all those boxes however is the one founded by Victoria Smith â SFGIRLBYBAY. Victoria is based in San Francisco and her daily blog features cool images, fab product reviews, food, lifestyle stories itâs a magazine unto itself and I am addicted.
I donât think anyone until they get into it realizes just how long it takes to compose a blog. Figuring out editorial content, taking images, composing the whole thing, takes enormous amounts of time and dedication so I take my hat off to Victoria for writing a blog that compels and has you coming back for more. I read www.sfgirlbybay.com daily, over breakfast at the computer eating Gwyneth Paltrow’s wheat free amazing granola every morning and I love it.
Victoria very kindly took some time out from her busy schedule to answer some questions like her coolest design sources, tips for rooms, favourite colour combos and Iâm totally inspired and checking them out!
Thank you Victoria
Favorite home furnishings store?
I am a big fan of DwellStudio. I feel like their furnishings are well-made and the style is timeless enough to fit a lot of different genres. They can go very traditional or completely eclectic. For more unusual pieces I love AnthropologieÂ and H.D. Buttercup.Â And the flea market is my go-to source for just about everything.
Tips for creating an unforgettable room.
I try to stay away from cookie-cutter catalog looks. I’m not a fan of decor that doesn’t reflect one’s personality or is too expected. I think designing is an extension of your personality. It’s the place I know I feel most comfortable and feeds my creativity. I decorate using personal elements, collections and artwork that really mean something to me, or makes me feel happy when I see them. If you do that, you’ll build a room that you want to spend time in. So, I think if you follow your heart then you’ll represent yourself uniquely — since there’s only one you.
How would you describe your style?
It’s super eclectic – I like to call it ‘bohemian modern’. It’s a mix of vintage, industrial and classic mid-century modern pieces, with colorful textiles to warm up the more austere furnishings. With the exception of a few key pieces like my black leather sofa and some custom book shelving I had built by Michael Woo, most of my home is decorated with flea market finds.
Youâre go to source for lighting and rugs?
Schoolhouse Electric has great lighting fixtures, and Tom Dixon pendant lamps are quite swoony! I’m really into Moroccan Boucherite rugs right now and there’s a few online sources I like: BeldiÂ and Love Adorned.
Favourite colour combinations?
I really like black and white with fresh pops of pink. And then again, I love charcoal gray and orange together. I think it depends entirely on my mood, and the time of year.
What has been the greatest challenge or challenges so far in running your business ?
Time management. I pretty horrid at it to tell the truth.Â In an ideal world, I’m caught up a week or at least a few days in advance, but lately it’s been so busy, I’m posting for the following day. So I am either editing my own photos and adding graphics, or rounding up images from the internet and putting curations together. That could go on all day and I’m sometimes up until 10 or 11 doing posts. Of course, there’s time for tweeting and posting on Facebook in-between. And answering important emails as they pop up. Somewhere in there, I eat. Not very well. I’m working on that. Some days, I go out and photograph a house, or a new shop in the city. Sometimes, and it’s rare, I go out for lunch. I hired an assistant about a month ago, and that’s been a huge improvement.
The content on your blog feels very inspiring,Â never dampened. How do you keep that alive?
Thank you! I am constantly on the lookout for new material – – my life and my job intersect a lot, so if I’m out somewhere running errands for instance, I might see something that makes a great photo, or a shop that I should feature. Or an art opening, or an event I attend can become a blog post. It all overlaps. I have just a handful of contributors – they contribute a post every other week and they generally have a theme. Danielle Krysa from The Jealous Curator posts about amazing art and how to incorporate it into your home; Si Mazouz from French by Design shares gorgeous home tours from primarily international/European homes; Aaron de Simone from I Heart Luxe is one of the most popular pinners out there and he shares a curation of his favorite pins for the week; and Nathan Michael is a photographer from Chicago who shares great photographic stories from his travels. I try to choose contributors that can offer something I can’t and perspectives I think the readers will enjoy. I’m horrible about keeping an Editorial Calendar. I am hoping since I can’t seem to remember when a certain themed post is supposed to run, the readers can’t remember either. But I do have regular features I do stick to like Tuesday’s Girl is all about fashion, and I run Unexpected Guests, Thoughts For a Friday, and Separated at Birth among others.
Where do you see blogs going in the future?
That’s a good question. I think some see them going away in the wake of Pinterest, but I think Pinterest only opens up opportunities for bloggers to share their design sensibilities and find like-minded followers.Â I hope blogs stay around – I feel like they are just getting started in many ways and they are finally getting some much-deserved recognition and respect from the media and advertisers. With many magazines closing (at least in the States) blogs are a great creative, inspiring resource for people interested in all kinds of design. I think they’re will be more sponsored posts, but if bloggers work with advertisers that are a good fit for their brand and readership, and blogs really think creatively about how they share brands you can create some really unique content because you have a budget that allows for great styling and photography.
Some images below from Victoriaâs pinterest boards below which are certainly worth following as well as her wonderful blog:

domain: artistic