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We remember when Siggi Hilmarsson’s skyr (pronounced SKEER), Icelandic-style strained yogurt, first appeared on the shelves of Murray’s Cheese in Greenwich Village.
Hailing from Iceland, the transplanted New Yorker found the yogurts in the U.S. too sweet and not thick enough—even the Greek-style yogurts. So in 2004 he started to make his own, in his kitchen. Today, Siggi’s skyr is available nationally, to the delight of many.
This is not bargain yogurt. It’s even pricier than Greek brands—and it’s thicker than Greek yogurt as well. The reason is, more milk is required to produce the same quantity. You get what you pay for.
Greek-style yogurt is thicker than American-style yogurt because more water is strained out of the whey—it’s triple strained. But skyr is drained even more. Think of it as quadruple-strained yogurt. One cup of Siggi’s skyr requires four times more milk than a typical American brand.
The result is so thick that a spoon stands up straight in the cup; yet it has 0% fat (some flavors are lowfat, 2%). The concentration of milk also delivers more calcium and protein.
A bowl of Siggi’s skyr with pomegranate arils. Photo courtesy Siggi’s Dairy.
IS SKYR YOGURT OR CHEESE?
In Iceland, skyr is typically fat-free because all the cream has been removed to make butter.
If you look for information on skyr, you may find it referred to as a cheese. So is it yogurt or cheese? It depends on the recipe of the individual producer.
The recipe arrived in Iceland from Norway in the Middle Ages. It most likely was originally made as a cheese, with rennet. These days, some ism some isn’t. Siggi’s is yogurt.
The difference between a cultured dairy product, such as sour cream or yogurt, and a fresh cheese that looks just like it, such as fromage blanc or quark, is the addition of a coagulant, such as rennet.
With cottage cheese and ricotta, you can see the curds. With fromage blanc and quark (and most other cheeses), you can’t, because of the particular recipe. You also can’t tell the difference by tasting it. The textures of sour cream, yogurt, fromage blanc and quark are very similar.
Don’t confuse these fresh cheeses with yogurt cheese like labneh.
Regular yogurt is made by combining milk with live cultures. It is available plain and flavored, made from whole milk (5% fat), lowfat (1%) and fat-free (0%).
Greek yogurt follows the same recipe, but is triple strained, removing a portion of by the whey. This creates a thicker yogurt that is higher in protein. It may or may not be tangier than regular yogurt, depending on the processes of the particular brand.
Skyr, Icelandic yogurt, is even thicker than Greek yogurt. Think of it as quadruple-strained. It is made from skim milk (0%)—the cream is skimmed off to make butter. In Iceland it is often made from raw milk, which is not legal in the U.S. for fresh dairy products.
THE DIFFERENCE IN YOGURT
The more concentrated (strained) a style of yogurt is, the costlier it will be because it contains more milk and less water.
Check out our Yogurt Glossary for much more on the different types of yogurt.

domain: food


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According to a story by Colin Moynihan in the New York Times titled "Former Assistant to Jasper Johns Pleads Guilty in Thefts of Artworks," the culprit confessed in federal court to absconding with millions in artworks from his renowned employer.
As Moynihan wrote: "The assistant, James Meyer, was indicted in 2013 on charges connected to a scheme that prosecutors said involved the theft of at least 22 artworks over about six years. Mr. Meyer removed the works from Mr. Johns's studio in Sharon, Conn., prosecutors said, and delivered them to an art gallery in Lower Manhattan, where they were sold for about $6.5 million."
Johns, 84, who ranks among the world's most influential artists, first made his mark in the 1950s with lushly colored and textured paintings of maps, targets and the American flag. His emotionally enigmatic work was one of the foundations of the pop art movement.
Meyer, who worked for Johns for 27 years, stole works from a studio print drawer, claiming they were gifts when he sold them. According to Moynihan's story, the former studio helper may have to return $4 million and spend three years or so in prison.

domain: news


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Having cabbages in your kitchen is a sign of good health for you and your family. But is it the same for your pet? Can bearded dragons eat cabbage too? Well to answer your question right away, yes! As a matter of fact, many experts claim it to be one of the best ingredients for a salad.
Cabbage is one healthy leafy vegetable with loads of health benefits for both humans and lizard reptiles. But of course, our body mechanisms differ from theirs and so bearded dragons have some concerns regarding the cabbage diet unlike us. For instance, the types of cabbage really matter to these small reptiles as the health benefits differ for each different type of cabbage. Primarily, there are five types. Let us introduce you to its various types and their respective nutritious characteristics.
5 Various Types Of Cabbage
The five types of cabbage differ greatly in nutrients and hence impart different effects on the beardies. These include Bok Choy, Napa, Red Cabbage, Green Cabbage, and Savoy. Each of the cabbage types has different nutrient values and henceforth different benefits.
First on the list is Bok Choy cabbage, this is because it is the best and most recommended of all cabbage types. The characteristic that marks it top on the list is its highest calcium-to-phosphorus ratio. A sufficient amount of calcium does not allow phosphorus to cause a calcium deficiency which it normally does due to its calcium-absorbing ability. It also contains fiber and other minerals and vitamins. Moreover, it is low in fats and sugars to form an overall healthy meal. Hence, it is the most ideal cabbage for a beardie.
Napa is the second on the list and is also known as the Chinese cabbage. It also contains a good amount of calcium compared to the rest of the cabbage types that are discussed below. Its leaves are similar to lettuce and it has a milder and sweeter flavor. In addition to this, it also contains Vitamin C, Vitamin K, and Vitamin A. There are almost no fats in this veggie but a very high amount of fiber. However, Napa contains a high level of goitrogen and so it should not be given very often.
Red cabbage has a lower amount of fiber as compared to the others and more importantly, it has a lower level of calcium than Bok choy and Napa. Yet, it is considered one of the best options. It has all the nutrients including vitamins and minerals. Vitamin A aids in enhancing vision, Vitamin C improves the immune system, and Vitamin K helps in blood formation. Moreover, it contains magnesium, potassium, and manganese.
There aren’t many differences between red and green cabbage. They both have almost similar amounts of calcium and also have low fats and sugars like all the cabbage types. It is also high in fiber, vitamins, and minerals. However, green cabbage is known for containing the highest levels of goitrogens, therefore it’s only best if you feed this type of cabbage only sparingly unlike the other recommended types.
At last, we have Savoy! The reason why this cabbage type is listed in the end is that it is the least recommended. Savoy is an unpopular type of cabbage when it comes to the diet of a beardie. It does not have the ideal calcium-to-phosphorus ratio because of its higher phosphorus and lower calcium levels. As a result, regular intake of savoy may cause metabolic bone disorder due to increased calcium absorption by phosphorus.
Also, savoy has a high level of Vitamin A and might cause Vitamin A toxicity with other Vitamin A-rich foods. This type is also high in acid and low in Vitamin C and K; therefore, it is best to feed Savoy rarely or not at all. Furthermore, you may also like to read Can Bearded Dragons Eat Cauliflower.
Nutrition Chart For All Cabbage Types
We have described each type of cabbage for you to decide which one should be in your bearded dragon’s salad. Here are the actual figures for you to judge the authenticity of our recommendation. These values are for 100g of cabbage.
|Energy||13 Kcal||16 Kcal||15 Kcal||31 Kcal||27 Kcal|
|Water||95.32 g||94.39 g||94.3 g||90.39 g||91 g|
|Protein||1.5 g||1.2 g||1.1 g||1.4 g||2 g|
|Fat||0.2 g||0.2 g||0.5 g||0.16 g||0.1 g|
|Carbohydrate||2.18 g||3.3 g||2.4 g||7.37 g||6.1 g|
|Sugars||2.18 g||1.14 g||1.06 g||3.83 g||2.27 g|
|Fiber||1 g||1.2 g||1.6 g||2.1 g||1.2 g|
|Phosphorus||37 mg||29 mg||24 g||30 mg||42 mg|
|Calcium||105 mg||77 mg||33 mg||4g mg||35 mg|
Benefits & Downsides Of Cabbages
Overall, every type of cabbage has common benefits. Even Though one type may be healthier than the other, the overall benefits are commendable for a bearded dragon’s diet. Firstly, cabbage has the best calcium-to-phosphorus ratio as compared to other foods. It is a rich source of calcium and therefore strengthens the bones and teeth of young and adult beardies.
Secondly, these leafy vegetables are rich in Vitamin C. It helps in strengthening the immune system, healing wounds, growth, and also the repair of body tissues. Thirdly, cabbage contains Vitamin A in good amounts, so beardies feeding on cabbage develop better eyesight and are protected from night blindness. In addition, Vitamin K is also present for the formation of red blood cells, regulation of calcium levels, and bone metabolism.
Furthermore, the high amount of fiber helps in better bowel movement and a healthier digestive tract. Lastly, the wonder vegetable is extremely low in fats, cholesterol, and sugars. Therefore, your beloved pet will never face obesity or any other related diseases such as heart problems, kidney issues, and liver disorders.
Keeping the numerous health benefits of cabbage in mind, the downsides of cabbage are almost non-existent. However, they must be known by the caretaker of the beardie.
Firstly, cabbage contains a lot of water. Its water-retaining characteristic can be dangerous for lizards. Too much water can disturb the stomach and the digestive system. Excess water causes diarrhea and other digestive disturbances. Next, cabbage should be washed very thoroughly as fresh cabbage may contain dirt. The dirt may contain harmful substances.
Lastly, the savoy type of cabbage causes Vitamin A toxicity and can prove to be too acidic for your pet. Also, the high amount of fiber in cabbages can be excessive and cause digestive problems too. While having all these disadvantages, cabbage is overall a healthy vegetable if it’s the right type and is fed in appropriate amounts.
How Often Should They Eat Cabbage
Fortunately, you can feed cabbages in more quantities to bearded dragons as compared to other meals, but how often can bearded dragons eat cabbage? As per most experts, you can feed your cabbage once a week to your dragon. It is healthy to feed cabbage every week and depending upon your beardie, you can even feed them twice a week.
Can They Eat Cabbage Leaves
It is often asked if beardies can eat the cabbage leaves, well yes! It is safe to gobble up cabbage leaves for these lizards. Make sure to wash the leaves thoroughly, as it contains the most dirt and soil. Also, chop the leaves into small pieces before feeding them to your pet. The leaves have a slippery smooth texture and so can easily choke the Beardie.
Conclusively, bearded dragons can eat cabbage safely and benefit greatly from calcium, vitamin C, fiber, and other nutrients. Bok Choy, Napa, and red cabbage are highly recommended amongst all types of cabbage. Whereas green cabbage is fine too, Savoy should be avoided. Moreover, it contains a high water content and so should be fed once or twice a week. Make sure to wash the cabbage and chop the leaves before feeding it to your pet.
Frequently Asked Questions
Can bearded dragons eat cabbage stalks?
No, cabbage stalks should not be fed to beardies. It is hard and difficult to chew and digest by young beardies and even most adult bearded dragons. Plus, the stalk can choke the throat too. It is best to feed the main part of the cabbage which has the most nutritional value.
Can bearded dragons eat Purple Cabbage?
Purple cabbage is the other name for red cabbage. It can be safely fed to all beardies. It has a fair calcium-to-phosphorus ratio and is rich in vitamin C. It also contains minerals and other nutrients in moderate amounts.
Can Bearded dragons eat cooked cabbage?
It is best to feed only raw cabbage to pets. Cooked cabbage retains water and so adds to the already high-water content. The high-water value can cause diarrhea and other stomach disturbances. Also, the process of cooking decreases the nutritional value of vegetables, so there’s no point in putting in more effort when it’s only leading to unhealthy outcomes.
What other vegetables can a bearded dragon eat besides cabbage?
There are some other healthy and nutritious leafy vegetables for beardies too. These include Watercress, Turnip Greens, Dandelion Greens, Collards, and Grape Leaves. All of these are high in nutrition and have little or no harmful effects.
Emma is a pet enthusiast, and her way with words makes her an expressive writer. Her interests lie in healthcare and planning nutrition for various pets. She has two girls, and she’s passing her passion to them through occasional volunteer projects in the small neighborhood zoo. Emma joined our team as an enthusiast and has added more years to her experience by researching more about various creatures!


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IN THE NEWS: VA HOSPITAL WRONGFUL DEATH VERDICT INCREASED AFTER-THE-FACT
It took nearly a decade, but Wilma Coolidge has finally found justice on behalf of her brother Howard Southard, who died at the VA Medical Center in Buffalo, NY. This medical malpractice case revolved around a stent graft that was improperly installed in one of Southard’s arteries causing loss of blood flow to his kidneys. Southard was hospitalized for 118 days and endured more than fifty surgical procedures.
Originally awarded $2.1 million, the wrongful death verdict was recently raised to $3.9 million, based on the extended period of pain and suffering that resulted from errors committed by Southard’s surgeons and by the mechanical device they installed.
The news coverage of the verdict mentioned that the lawsuit led to a seventeen-day trial, pitting one medical expert against another. In cases of medical malpractice or neglect, an experienced attorney can be an invaluable asset in determining the appropriate course of action, as we well know at Ramey & Hailey Law. Because the provision of medical care is a highly technical and specialized field, expert testimony is often required to establish the connection between the actions of the medical practitioner and the resulting injury.
Howard Southard’s death represents a tragic tale of suffering, yet serves as an example that, with the right legal advice, survivors can gain compensation through the court system.


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Use everyday materials to design your own Halloween masks in just a few hours.
Your own backyard is the perfect setting for a creepy haunted maze that will give your friends a delightful fright.
Make plans this Halloween to take a vacation and visit some of the spookiest destinations in the country. Many of these areas have a tragic past, which adds to the chances of them being haunted and creating a spooky feeling among those who tour the area.
The child remains hospitalized in critical but stable condition
A sheriff’s investigation is under way after two trick-or-treaters reported receiving tampered candy Thursday night in the same subdivision in northern Nevada.
ZOPPÉ “An Italian Family Circus” returns to The Smith Center’s Symphony Park on Halloween for a four-day run. The up close show was so popular last year, they were asked to bring it back again.
Going trick-or-treating with the kids this Halloween? Elizabeth Bailey RN and Dr. Rebecca Bailey’s have some helpful tips for ensuring a safe and fun night out in the neighborhood.
Choose your favorite Halloween candy!
Haunted houses can be lots of fun, but visiting local haunting sights can be even more fun. Be sure to visit these ghostly spots when the sun goes down.
The fall season brings us the three-month stretch of holiday celebrations and to kick it all is the spookiest of them all – Halloween. Here are the five must-haves for your haunted house.

domain: entertainment


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A key advantage of nanomaterials for biomedical applications is their ability to feature multiple small reporter groups (multimodality), or combinations of reporter groups and therapeutic agents (multifunctionality), while being targeted to cell surface receptors. Here a facile combination of techniques for the syntheses of multimodal, targeted nanoparticles (NPs) is presented, whereby heat-induced-radiolabeling (HIR) labels NPs with radiometals and so-called click chemistry is used to attach bioactive groups to the NP surface. Click-reactive alkyne or azide groups were first attached to the nonradioactive clinical Feraheme (FH) NPs. Resulting “Alkyne-FH” and “Azide-FH” intermediates, like the parent NP, tolerated 89Zr labeling by the HIR method previously described. Subsequently, biomolecules were quickly conjugated to the radioactive NPs by either copper-catalyzed or copper-free click reactions with high efficiency. Synthesis of the Alkyne-FH or Azide-FH intermediates, followed by HIR and then by click reactions for biomolecule attachment, provides a simple and potentially general path for the synthesis of multimodal, multifunctional, and targeted NPs for biomedical applications.
Citation: Yuan H, Wilks MQ, El Fakhri G, Normandin MD, Kaittanis C, Josephson L (2017) Heat-induced-radiolabeling and click chemistry: A powerful combination for generating multifunctional nanomaterials. PLoS ONE 12(2): e0172722. https://doi.org/10.1371/journal.pone.0172722
Editor: Michael K. Chan, The Chinese University of Hong Kong, HONG KONG
Received: December 12, 2016; Accepted: February 8, 2017; Published: February 22, 2017
Copyright: © 2017 Yuan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: Support was provided by the NIH R01 EB017699 (LJ), the Prostate Cancer Foundation (CK), Alex’s Lemonade Stand Foundation (CK), NIH T32EB013180, NIH R01MH100350 (MN). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: (BimC4A)3, tripotassium 5,5′,5′′-[2,2′,2′′-nitrilotris(methylene)tris(1H-benzimidazole-2,1-diyl)]tripentanoate hydrate); 89Zr-Cy5.5-Alkyne-FH, 89Zr-labeled Cy5.5 fluorescent FH NPs through Alkyne-FH copper catalyzed click chemistry; 89Zr-Cy5.5-Azide-FH, 89Zr-labeled Cy5.5 fluorescent FH NPs through Azide-FH copperless click chemistry; 89Zr-Cy5.5-Protamine-FH, 89Zr-labeled Protamine modified FH NPs; 89Zr-Folate-FH, 89Zr-labeled folate modified FH NPs; 89Zr-RGD-FH, 89Zr-labeled cRGD peptide modified FH NPs; Alkyne-FH, alkyne modified FH NPs; Azide-FH, azide modified FH NPs; CMD, carboxymethyl dextran; Cy5.5-Alkyne-FH, Cy5.5 fluorescent FH NPs through Alkyne-FH copper catalyzed click chemistry; Cy5.5-Azide-FH, Cy5.5 fluorescent FH NPs through Azide-FH copperless click chemistry; Cy5.5, cyanine 5.5 fluorochrome; Cy5.5-Protamine-FH, Protamine modified FH NPs; DBCO, dibenzocyclooctyne; DFO, deferoxamine; DMSO, dimethyl sulfoxide; DPBS, Dulbecco’s phosphate buffered saline without calcium and magnesium; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; FBS, fetal bovine serum; FH, Feraheme; Folate-FH, folate modified FH NPs; HIR, Heat-Induced Radiolabeling; HOBT, 1-hydroxybenzotriazole; ICP-MS, inductively coupled plasma mass spectrometry; MES, 2-(N-morpholino)ethanesulfonic acid; MRI, magnetic resonance imaging; NPs, nanoparticles; PD-10, desalting column with Sephadex G-25 resin; PET, positron emission tomography; RCP, radiochemical purity; RCY, radiochemical yield; RGD-FH, cRGD peptide modified FH NPs; SPECT, single photon emission computed tomography; TFA, trifluoroacetic acid
The value of multimodal (detectable more than one modality) or multifunctional (therapeutic and diagnostic) nanomaterials has been discussed and been the subject of recent comprehensive reviews.[2, 3] Here we provide a facile and general approach to the design of targeted, theranostic nanoparticles (NPs) whereby Feraheme (FH) nanoparticles (NPs) undergo a heat induced radiolabeling (HIR) for detection by nuclear techniques, followed by biomolecule attachment to the NP surface using mild click chemistry reactions. The HIR technique employs heat (120°C, 2 hr) to bind cations to the surface of 5 nm iron oxides.[4, 5]
Though one of several chelator-free methods for radiolabeling nanomaterials, HIR is unique in its ability to label a NP drug with any of three cations used in PET or SPECT (64Cu2+, 111In3+ or 89Zr4+).[4, 5] These procedures yield MRI detectable FH NPs that can be labeled with therapeutic radiometals like 111In3+ (a low dose SPECT isotope and high dose therapeutic ) or 67Cu2+ . In addition, FH (1) has been shown to bind a wide range of non-radioactive metals by ICP-MS . Given the broad availability of Feraheme (FH) NPs, the radiocation flexibility of the HIR reaction, and the many commercially available click reagents, we reasoned that HIR and click chemistry surface functionalization might make a useful and general combination for the synthesis of multimodal (radioactive, magnetic, fluorescent), targeted NPs if the azide and alkyne groups survived the HIR reaction.
We therefore combined HIR and click chemistry surface functionalization as summarized in Fig 1 and provided in detail in Figs 2–4. The starting FH NP (Fig 1, upper left) has a 5 nm core of superparamagnetic iron oxide (black central ball) consisting of approximately 5874 Fe/NP (manufacturer’s package insert). Surrounding the core is a thick (10 nm) coating of polymeric carboxymethyl dextran (CMD, light blue lines), so the NP has an overall size of 20–25 nm. Previously, this starting FH NP was subjected to the HIR reaction, shown here as the creation of a radioactive FH (red ball in figures).[4, 5] Here we used FH NPs for the synthesis of two non-radioactive click reactive intermediates; these were the azide functionalized NP (Azide-FH, 4) or an alkyne functionalized NP (Alkyne-FH, 5). The NPs, Azide-FH (4) and Alkyne-FH, (5), were then reacted with commercially available, click reactive Cy5.5 fluorochromes, to demonstrate the presence reactive azides and alkynes on the NP surface and provide a path to nonradioactive but magnetic and fluorescent NPs for future studies. Finally, Azide-FH (4) and Alkyne-FH (5) were subjected to HIR; our demonstration uses 89Zr and the number of reactive azides and alkynes on the resulting 89Zr-Azide-FH, (89Zr-4) and 89Zr-Alkyne-FH (89Zr-5) NPs were compared with the number on the precursor (Azide-FH, 4 and Alkyne-FH, 5) NPs to determine whether azide and alkyne groups survived the HIR reaction. Since the alkynes and azides survived HIR (see below and Table 1), the 89Zr-Azide-FH (89Zr-4) and 89Zr-Alkyne-FH (89Zr-5) NPs were then reacted with click reactive targeting molecules, yielding 89Zr-Folate-FH (89Zr-11), 89Zr-RGD-FH (89Zr-14), and 89Zr-Cy5.5-Protamine-FH (89Zr-16) as shown in Figs 3–4.
Our previous HIR of FH NPs (top) yielded non-surface functionalized, radioactive NPs (red core). The alkyne and azide functionalized FH intermediates were synthesized (Azide-FH, 4 and Alkyne-FH, 5) and labeled by HIR reaction, to yield 89Zr-Azide-FH (89Zr-4) and 89Zr-Alkyne-FH (89Zr-5). Imaging detection modalities for the NPs are in bold. NPs targeted to folate receptors (89Zr-Folate-FH, 89Zr-11), integrins (RGD-FH, 14) or NPs with protamines (89Zr-Cy5.5-Protamine-FH, 89Zr-16) were then synthesized. Detailed synthetic schemes are given in Figs 2–4.
a) Using FH (1), the Azide-FH (4) and Alkyne-FH (5) were synthesized. Portions of Azide-FH (4) and Alkyne-FH (5) were then radiolabeled by HIR, yielding 89Zr-Azide-FH (89Zr-4) and 89Zr-Alkyne-FH (89Zr-5). To determine reactive azide or reactive alkynes, NPs were reacted with the appropriate click reactive Cy5.5 fluorochromes, with Cy5.5s shown as the yellow stars of Fig 1. After removal of the unreacted Cy5.5s (DBCO-Cy5.5, 6 or Azide-Cy5.5, 7), the number of Cy5.5’s per NP was determined from absorption spectra examples of which are shown in Fig 2b–2e. Controls for covalent binding were a reaction of FH (1) and DBCO-Cy5.5 (6) and a reaction of Azide-FH (4) and DBCO-Cy5.5 (6) preoccupied with DBCO-NH2. Values in parentheses are the numbers of reactive groups per NP with values summarized in Table 1.
Non-radioactive Alkyne-FH (5) or radioactive Alkyne-FH (89Zr-Alkyne-FH, 89Zr-5) was reacted with the Azide-Cy5.5 (7) before reaction with an azide bearing folate (10). After reaction with Azide-Cy5.5 (7), the number of folate groups, p, on the NP was determined as reactive alkynes before and after folate reaction; that is as 89Zr-Cy5.5-Alkyne-FH (89Zr-9) (m) minus the number on 89Zr-Cy5.5-Folate-FH (89Zr-12) (q).
89Zr-Azide-FH (89Zr-4) or Azide-FH (4) was reacted with DBCO-PEG4-RGD (13) and the number of RGDs per NP on 89Zr-RGD-FH (89Zr-14) or RGD-FH (14) was determined by the “Before and After Fluorochrome Reaction and Subtraction Method”. DBCO-Protamine-Cy5.5 (15) has a C-terminal Cy5.5 was synthesized (see S2 File), and this compound was used to synthesize 89Zr-Cy5.5-Protamine-FH (89Zr-16) or Cy5.5-Protamine-FH (16).
Materials and methods
All water was Chelex treated. Reactivial was from Wheaton (0.3 ml, W986253NG). Chemicals/vendors were Feraheme/AMAG Pharmaceuticals; 89Zr oxalate/Perkin Elmer @ 0.15GBq/nmol in 1M oxalate; desferoxamine/Sigma (D9533); Azido-PEG3-Amine (2), Alkyne-PEG4-Amine (3), DBCO-Cy5.5 (6), DBCO-PEG4-NHS ester (A102P-10), and Azide-Cy5.5 (7)/all were from Click Chemistry Tools; Azide-Folate (10)/Barry & Associates; (BimC4A)3 (696854-250MG, tripotassium 5,5′,5′′-[2,2′,2′′-nitrilotris(methylene)tris(1H-benzimidazole-2,1-diyl)]tripentanoate hydrate)/Sigma-Aldrich. HOBT/#H0468-TCI; EDC/ #22980-Thermo Scientific; Protamine/APP Pharmaceuticals; Cy5.5-amine/470C0-Lumiprobe. All the other solvents and chemicals were from Sigma-Aldrich. Elution profiles from PD-10 columns (GE Healthcare) were analyzed with a Perkin Elmer Wizard 2480 Gamma Counter. DBCO-PEG4-RGD (13, Fig 4) was synthesized as described . Relaxation times were determined on a Bruker Mini SPEC MQ20, and particle sizes determined on a Malvern Instruments, ZetaSizer Nano Series, Nano-ZS. UV spectra were on Thermo Scientific, Evolution 300 UV-VIS. NPs were washed and concentrated by ultrafiltration (50 kDa Amicon ultrafilters and centrifugation). HPLC (Varian ProStar detector and delivery modules) employed an eluant A (0.1% TFA /water) and eluant B (0.1% TFA in 9.9% water in acetonitrile). Iron was quantified by absorbance at 450 nm using dilutions of 30 mg Fe/ mL Feraheme stock drug for a standard curve.
Syntheses of Azide-FH (4) and Alkyne-FH (5), see Fig 2a
FH (1, 400 μL, 12 mg Fe) was transferred to 0.1 M, pH 6.0–6.4 MES by use of a PD-10 column equilibrated with this buffer. For Azide-FH (4), FH (1, 12 mg Fe in 2.1 mL MES buffer), HOBT (10.55 mg, 0.078 mmol), and EDC (55.27 mg, 0.288 mmol) were incubated (room temperature, 20 min). A DMSO solution of Azido-PEG3-Amine (2, 1 M, 80 μL) was added (50°C, 2 h). Low molecular weight materials were removed and NP concentrated by ultrafiltration. The NP was by PD-10 (chelex treated water) purified and concentrated by ultrafiltration to obtain a stock solution (5mg Fe/mL in chelex water (2.2 mL)) for the copperless click reaction. For Alkyne-FH (5), a solution of Alkyne-PEG4-Amine in DMSO (3, 0.1 M, 800 μL) was reacted with FH (1), purified and concentrated as for Azide-FH (4) and also obtained as a 5 mg Fe/mL in chelex water.
Demonstration of covalent click reaction, see Fig 2
The azide groups on the Azide-FH NP (4) (0.32 mg Fe in water 200 μL) was blocked by addition of DBCO-amine (Click Chemistry Tools, A103, 3.2 μL, 20 mM in DMSO) in a 0.3 mL glass vial (room temperature, 1 h). NPs were DPBS PD-10 purified and concentrated by ultrafiltration. DPBS (200 μL) and DBCO-Cy5.5 (6, 6.85 μL, 6.78 mM in DMSO) were added to the retentate and incubated (room temperature, 1 hr). NPs were PD-10 purified, concentrated by ultrafiltration and the number of Cy5.5/NP (1.3) determined by absorbance (Fig 2d). A second control was FH (1) (0.32 mg Fe) in chelexed water (200 μL) reacted with DBCO-Cy5.5 (6, 6.85 μL, 6.78 mM in DMSO, room temperature, 1 h). The NP was purified and Cy5.5/NP (<1) determined as above (Fig 2e).
Determination of azides on Azide-FH (4) by reaction with DBCO-Cy5.5 (6), Fig 2
Azide-FH (4, 40 μL, 0.2 mg Fe), DBCO-Cy5.5 (6, 6.85 μL, 6.78 mM in DMSO), and DPBS (160 μL) were incubated (room temperature, 1 h) followed by a PD-10 (in PBS) purification. The NP Cy5.5-Azide-FH (8) was concentrated by ultrafiltration.
Determination of alkynes on Alkyne-FH (5) by reaction with Azide-Cy5.5 (7)
Alkyne-FH (5) (40 μL, 0.2 mg Fe), Azide-Cy5.5 (7, 11 μL, 4.225 mM in DMSO), and Cu-(BimC4A)3-ascorbate catalyst stock solution (5 μL, see S1 File) and DPBS (160 μL) were incubated at room temperature for 15 min followed by a PD-10 (in PBS) purification. The NP Cy5.5-Alkyne-FH (9) was concentrated by ultrafiltration.
Syntheses of 89Zr-Azide-FH (89Zr-4) and 89Zr-Alkyne-FH (89Zr-5), see Fig 2a
In a 0.3 mL glass reactivial with magnetic stir bar was placed 89Zr-oxalate (0.744 mCi, 1 M oxalic acid, 42 μL) with chelex water (50 μL) and the mixture was brought to pH 8–9 with 1M Na2CO3. Azide-FH (4, 120 μL, 0.6 mg Fe) was added and pH was adjusted. The mixture was heated (120°C, 2 hr, silicon oil bath) and then cooled (ice bath, 15 min, stirring). DFO (3 μL, 20 mM, pH 7.5) was added to remove loosely bound 89Zr (15 min). The 89Zr-Azide-FH (89Zr-4) was purified by PD-10 (PBS) and concentrated by ultrafiltration, yielding 89Zr-Azide-FH (89Zr-4) (1.3 mg Fe/ml) in DPBS (pH 7.4) for copperless click reactions. The product was analyzed for purity by PD-10 in PBS (RCP > 99%, S1 Fig). RCY: 0.672 mCi, 90%. Specific activity: 62.8 mCi/mmol Fe. Synthesis of 89Zr-Alkyne-FH (89Zr-5) was as for 89Zr-Azide-FH (89Zr-4), yielding 89Zr-Alkyne-FH (89Zr-5) (1.3 mg Fe/ml) in DPBS (pH 7.4) for copper mediated click reactions. 89Zr-Alkyne-FH (89Zr-5) was analyzed by PD-10 (RCP >99%, S2 Fig). RCY: 0.527 mCi, 87.8%; Specific activity: 49.3 mCi/mmol Fe.
Reaction of 89Zr-Azide-FH (89Zr-4) and 89Zr-Alkyne-FH (89Zr-5) with Cy5.5s, Fig 2
To a 0.3 mL glass reactivial with magnetic stir bar were mixed 89Zr-Azide-FH (89Zr-4) (0.2 mg Fe, 150μl, 0.208 mCi) and DBCO-Cy5.5 (6, 6.85 μL, 6.78 mM in DMSO). The mixture was incubated (room temperature, 1 hr) with 89Zr-Cy5.5-Azide-FH (89Zr-8) obtained by PD-10 purification and ultrafiltration. 89Zr-Cy5.5-Azide-FH (89Zr-8) was analyzed by PD-10 gel-filtration (S3 Fig) and formulated in DPBS (pH 7.4) (200 μL). RCY: 0.17 mCi, 81.7%; Specific activity: 47.6 mCi/mmol Fe; RCP >99% (S3 Fig). The ratio of Cy5.5/FHNP (14.5) (Table 1) indicated reactive azides on the starting 89Zr-Azide-FH (89Zr-4). Similarly, 89Zr-Alkyne-FH (89Zr-5) (0.16 mg Fe, 117 μL, 0.135 mCi), Azide-Cy5.5 (7, 11 μL, 4.225 mM in DMSO), and Cu-(BimC4A)3-Ascorbate stock solution (5 μL, see S1 File) was incubated under magnetic stirring (room temperature, 15 min). 89Zr-Cy5.5-Alkyne-FH (89Zr-9) was purified, collected, and concentrated as for 89Zr-Cy5.5-Azide-FH (89Zr-8). The RCP was analyzed by PD-10 (RCP >99%, S4 Fig). RCY: 0.111 mCi, 82.2%. Specific activity: 38.9 mCi/mmol Fe. The ratio of Cy5.5/FHNP (12.2) (Table 1).
Syntheses of the folate functionalized FHs (Folate-FH, 11, 89Zr-Folate-FH, 89Zr-11, 89Zr-Cy5.5-Folate-FH, 89Zr-12), see Fig 3
Alkyne-FH (5, 0.2 mg Fe), Azide-Folate (10, 4.6 μL, 10 mM in DMSO), a Cu-(BimC4A)3-Ascorbate stock solution (5 μL, see S1 File) and 200 μL DPBS were incubated (room temperature, 15 min.) Folate-FH (11) was purified, collected, and concentrated as for 89Zr-Cy5.5-Azide-FH (89Zr-8). The ratio of folate/FHNP (10.5, Table 1) was calculated by determining the numbers of Cy5.5/NP before and after reaction of Azide-Folate (10) using Azide-Cy5.5 (7). 89Zr-Folate-FH (89Zr-11) was synthesized as for Folate-FH (11) but with 89Zr-Alkyne-FH (5). RCY: 0.111 mCi, 64.2%; Specific activity: 30.9 mCi/mmol Fe; RCP >99%. See S5 Fig. Ratio of folate/FHNP was 12.2, (Table 1). 89Zr-Folate-FH (89Zr-11) (0.2mg Fe, 150 μL DPBS, 0.173 mCi) with Azide-Cy5.5 (7, 11 μL, 4.225 mM in DMSO) by incubation at room temperature for 15 min using (BimC4A)3-Ascorbate stock solution (5 μL, see S1 File) as the catalyst. 89Zr-Cy5.5-Folate-FH (89Zr-12) was purified, collected, and concentrated as for 89Zr-Cy5.5-Azide-FH (89Zr-8). RCY: 0.119 mCi, 68.8%; Specific activity: 33.3 mCi/mmol Fe; RCP >99% (S6 Fig).
Synthesis of RGD functionalized FHs, (RGD-FH, 14), see Fig 4
Azide-FH (4, 0.2 mg Fe) and DBCO-PEG4-RGD (13, 5 μL, 15.6 mM in DMSO) was incubated under room temperature in chelexed water (200 μL) for 1 hr. The mixture was purified by PD-10 column (PBS) and concentrated by ultrafiltration. A ratio of RGD/FHNP (11.7, Table 1) was obtained by reaction with DBCO-Cy5.5 (6).
Synthesis of protamine functionalized FHs, (Cy5.5-Protamine-FH, 16 and 89Zr-Cy5.5-Protamine-FH, 89Zr-16), see Fig 4
For Cy5.5-Protamine-FH (16), to a solution of Azide-FH (4, 0.2 mg Fe, 40 μL) and DPBS (160 μL), was added DBCO-Protamine-Cy5.5 (15, 15 μL, 0.12 mM in chelexed water) under stirring. (Synthesis of DBCO-Protamine-Cy5.5 (15) is given in S2 File). The mixture was incubated (room temperature, 30 min). The mixture was loaded on a PBS preconditioned Sephadex G-50 column, eluted with PBS and concentrated by ultrafiltration. A ratio of Protamine/FHNP (3.4, Table 1) was obtained by UV absorbance of Cy5.5. The attachment of protamine was confirmed by a fluorescence increase after the trypsin digestion (S2 File), which cleaves the high arginine protamine and reduces fluorescence quenching.
For 89Zr-Cy5.5-Protamine-FH (89Zr-16), to a 0.3 mL glass reactivial with 89Zr-Azide-FH (89Zr-4) (0.5 mg Fe, 0.457 mCi) in DPBS (0.25 mL), was added the stock solution of DBCO-protamine-Cy5.5 (15, 25 μL, 0.12 mM in chelexed water). In order to get a more homogeneous payload distribution, the stock solution of DBCO-Protamine-Cy5.5 (15) was added under well stirring by fractional additions for 5 times with 5 μL every 3 min. After the complete addition of DBCO-Protamine-Cy5.5 (15), the mixture was further stirred under room temperature for 30 min. A DPBS conditioned Sephadex G-50 column was employed for purification. The product was collected and reconstituted in DPBS (1.4 mL) without centrifugation. The iron concentration (6.66 mM) and protamine/NP ratio (2.6, Table 1) were measured with UV absorbance. RCY: 0.387 mCi, 84.7%. RCP was analyzed by PD-10 SEC analysis (RCP > 99%, see S7 Fig). Specific activity: 43.2 mCi/mmol Fe.
Cell binding study of RGD-FH (14) with BT-20 cells (Fig 5)
(a) Flow cytometry histograms for the reaction of RGD-FH (14) and a control NP (b) at three NP concentrations (18 nM, 65 nM, and 155 nM) with BT-20 cells are shown. NP concentrations were determined from iron concentrations using the manufacture’s value of 5874 Fe’s per NP (AMAG Pharmaceuticals Package insert.) Relative fluorescence (c) from (a) and (b) versus NP concentration is shown. Relative fluorescence is the mean fluorescence of (a) minus the mean fluorescence of (b) divided by the fluorescence of unstained cells. (d) Uptake of the 89Zr-Folate-NP (89Zr-11) and a 89Zr-FH control NP is shown in at three NP concentrations. (e) Uptake was blocked by 1 μM folate (10) and was highly temperature dependent (f). (g) Uptake of 89Zr-Cy5.5-Protamine-NP (89Zr-16) at three different NP concentrations is shown. (h) 89Zr-Cy5.5-Protamine-FH (89Zr-16) uptake at 4°C and 37°C is shown (10 nM NPs were used). MDA-MB-231 cells were used for (d) through (h).
(125,000 cells/well) were plated in a 12-well plate on day 0. On day 1, media was aspirated and cells were washed with FBS-augmented DPBS (2% FBS). Cells were then incubated with 167 nM, 55.5 nM, or 18.5 nM nanoparticle suspended in DPBS (2% FBS). After 0.5 h, media was aspirated, cells were washed twice with DPBS (2% FBS), trypsinized and resuspended in DPBS, for measurement by single channel flow cytometry (BD-LSRII Cytometer).
Cell binding study of 89Zr-Folate-FH (89Zr-11) with MDA-MB-231 cells (Fig 5d–5f)
Cells were grown according to the supplier’s recommendation in a humidified incubator at 37°C, 5% CO2. Two hundred thousand cells were seeded on 6-well Costar plates (Corning, NY) in a 2 mL media, followed by overnight incubationat 37°C, 5% CO2. They were then treated with 1X PBS-diluted compound, and placed in the incubator for 6 h. Cells were washed three times with 1X PBS at 37°C, followed by a 10 min lysis in RIPA buffer. The solution was then transferred to cytometry tubes, and radioactivity was measured (Perkin Elmer Wizard 2480 Gamma Counter). To block endocytic uptakeof 89Zr-Folate-FH, the cells were incubated for1 h at 4°C with the NPs (final concentration = 3 nM). Control cells were incubated with HIR labeled FH,89Zr-FH NP (final concentration = 10 nM) for 2 h (37°C or 4°C). Alternatively, 89Zr-Folate-FH NPs (final concentration = 1 nM) were incubated with cells and folate ((10) @ 1 μM) for 1 h at 37°C.
Cell binding study of 89Zr-Cy5.5-Protamine-FH (89Zr-16) with MDA-MB-231 cells(Fig 5g and 5h)
For the uptake of the radiolabeled Protamine-FH compound, two hundred thousand cells were seeded on 6-well Costar plates (Corning, NY) in 2 mL media, followed by overnight incubation at 37°C, 5% CO2. They were then treated with 1X PBS-diluted compound, and placed in the incubator for 2 h. Subsequently, they were washed three times with 1X PBS, which was warmed at 37°C, followed by 10-minute lysis in the presence of RIPA buffer. Prevention of endocytic uptake was performed by placing the cells for 1 h at 4°C in the presence of the nanoparticles (final concentration = 10 nM). Cell-associated radioactivity was performed similar to the study investigating the uptake of radiolabeled-folate-FH.
Syntheses of azide and alkyne functionalized FH NPs
The carboxyl groups of carboxymethyl dextran (CMD) create a heat resistant bond between the iron oxide [11, 12] and provide sites for reaction with amines [13, 14]. So FH (1) was reacted with Azido-PEG3-Amine (2) or Alkyne-PEG4-Amine (3) using HOBT and EDC as shown in Fig 2a.
To demonstrate the presence of click reactive groups on the putative Azide-FH (4) or Alkyne-FH (5), NPs were reacted click reactive fluorochromes, DBCO-Cy5.5 (6) or Azide-Cy5.5 (7). (Structures are given in the Supplement, S8 Fig.) After size exclusion chromatography to remove unreacted fluorochrome, the concentration of Cy5.5 was determined spectrophotometrically (absorbance @ 675 nm, extinction coefficient = 250,000 M-1 cm-1), see Fig 2b and 2c. With the iron concentration determined and using the manufacturer’s figure of 5874 Fe per NP, there were 14.9 reactive azides based on the value 14.9 Cy5.5s/NP reactive per NP (Fig 2b). Similarly, there were 13.1 reactive alkynes per NP (Fig 2c). Values are summarized in Table 1.
To demonstrate that the association between Cy5.5 and NPs resulted from a click-based covalent reaction, and not adsorption of Cy5.5 to the NP surface two types of controls were run. First, the reaction between Azide-FH (4) and DBCO-Cy5.5 (6) was determined after incubation with DBCO-NH2 which reduced n from 14.3 to 1.3 (Fig 2d). Second, DBCO-Cy5.5 (6) was reacted with FH (1) and gave no detectable reaction (Fig 2e). As summarized in Table 1, FH can be derivatized with click reactive azide or alkyne groups and the numbers of reactive groups per NP determined by reaction with an appropriate Cy5.5 fluorochrome.
89Zr HIR of Azide-FH (4) and Alkyne-FH (5) and azide and alkyne’s tolerance of HIR proved by the reaction with the “clickable” Cy5.5 fluorochromes (which are compounds DBCO-Cy5.5 (6) and Azide-Cy5.5 (7) in Fig 2.)
The Azide-FH (4) and Alkyne-FH (5) were radiolabeled with 89Zr by HIR, yielding 89Zr-Azide-FH (89Zr-4) and 89Zr-Alkyne-FH (89Zr-5), which were then reacted with click reactive Cy5.5’s, see Fig 2a with results summarized in Table 1. (Sizes and relaxivities were determined only for non-radioactive NPs to avoid possible instrument contamination.) The reaction of 89Zr-Azide-FH (89Zr-4) and 89Zr-Alkyne-FH (89Zr-5) with Cy5.5 indicates the numbers of reactive groups per NP and provides a path radioactive, magnetic and fluorescent multimodal NPs for future use. Results summarized in Table 1 indicate FH can be functionalized with alkyne or azide that survive HIR. The reaction of 89Zr-Azide-FH (89Zr-4) or 89Zr-Alkyne-FH (89Zr-5) (or their non-radioactive counterparts) with targeting groups was therefore undertaken.
Click conjugation of biomolecule targeting groups on HIR and non-HIR FH NPs
To demonstrate the use of the HIR and click chemistry surface modification combination for the design of targeted NPs, we selected three well-studied targeting groups (folate, an RGD peptide, protamine) for study. The interaction of folate-bearing conjugates with the folate receptor has been used to target NIR fluorochromes [15, 16], radioisotopes [17–19], polymers  and NPs [21–23]. Similarly, the interaction of RGD peptides with RGD binding integrins [24–27] has been used to target a wide range of radioisotopes [28, 29], fluorochromes [30, 31], polymers , and NPs [30, 33]. RGD binding integrins are upregulated on angiogenic endothelial cells. Finally, high arginine peptides like protamine serve as a membrane translocation signals, inducing internalization of nucleic acids and NPs [34–37].
Folate attachment by a copper mediated click reaction.
We employed a copper mediated click reaction between the alkyne-functionalized FH’s (89Zr-Alkyne-FH, 89Zr-5 or Alkyne-FH, 5) and Azide-Folate (10), to obtain folate functionalized NPs (89Zr-Folate-FH, 89Zr-11 or Folate-FH, 11) as shown in Fig 3. To demonstrate the presence of folates on the NP surface, NPs were reacted with an azide-bearing Cy5.5 (7) before and after their reaction with azide-folate. The number of folates per NP, p, was then determined by subtraction, p = m—q, where m is the number of reactive alkynes before folate attachment and q is the number of reactive alkynes remaining after folate attachment (Table 1). Using this technique there were 12.2 reactive alkynes on 89Zr-Alkyne-FH (89Zr-5) and 12.2 folates on 89Zr-Cy5.5-Alkyne-FH (89Zr-9). We term this as the“Before and After Fluorochrome Reaction and Subtraction Method” for determination of targeting groups on NP surfaces.
RGD and protamine attachment by copperless click reactions.
To demonstrate a surface modification of 89Zr-Azide-FHs (89Zr-4) or Azide-FH (4) using a copperless click reaction, the NP was reacted with a DBCO-bearing RGD peptide (Fig 4), and the number of surface RGDs determined with the “Before and After Fluorochrome Reaction and Subtraction Method” to be 11.7 RGD’s per NP.
To obtain a protamine functionalized NPs, a protamine with an N-terminal DBCO and C-terminal Cy5.5 was synthesized. (Synthesis of DBCO-Protamine-Cy5.5 (15) is provided in S2 File). Unlike folate or RGD targeting molecules, only small numbers of protamines per NP could be attached without agglomeration (Fig 4). Hence the numbers of protamines per NP (2.6–3.4) was demonstrated directly from the numbers of Cy5.5s on NPs (Cy5.5-Protamine-FH, 16 or 89Zr-Cy5.5-Protamine-FH, 89Zr-16) as summarized in Table 1.
Bioactivity of surface functionalized NPs
To demonstrate that the surface functionalized NPs synthesized possess bioactive functional groups three experiments were conducted (Fig 5): (i) the binding of RGD functionalized NPs (RGD-FH, 14, Fig 4) on integrin expressing cells was examined using fluorescence (flow cytometry, Fig 5, Panel of RGD NP). (ii) the binding of HIR labeled, folate functionalized NPs (89Zr-Folate-FH, 89Zr-11, Fig 3) using folate receptor bearing cells was examined (Fig 5, Panel of Folate NP). (iii) The uptake of HIR labeled protamine functionalized NPs (89Zr-Cy5.5-Protamine-FH, 89Zr-16, Fig 4) was examined (Fig 5, Panel of Protamine NP).
A NP Cy5.5-Azide-FH (8) bearing 8.7 Cy5.5/NP s and second bearing 8.7 Cy5.5/NP and 18.8 RGDs/NP (RGD-Cy5.5-FH) were synthesized by reacting Azide-FH (4) with DBCO-Cy5.5 (6), or by reacting Cy5.5-Azide-FH (8) with DBCO-PEG4-RGD (13). The above fluorescent version of RGD bearing NP (RGD-Cy5.5-FH) and control NPs Cy5.5-Azide-FH (8) were incubated with BT-20 integrin-expressing cells [38, 39] and cell fluorescence examined by flow cytometry (Fig 5a and 5b). Fluorescence was higher for the RGD bearing RGD-FH (14) than the control NP at three NP concentrations, indicating NPs-bearing RGD peptides bound to cellular integrins (Fig 5c). The RGD peptides on RGD-FH (14) bind integrins expressed on BT-20 cells.
The interaction of 89Zr-Folate-FH (89Zr-11) (Fig 3, Folate/NP = 9.0) with folate receptor-bearing MDA-MA-231 cells [8, 40] was examined as shown Fig 5d–5f with HIR 89Zr-FH NP  as a control for nonspecific binding. NPs were used at three concentrations (Fig 5d), with far higher uptake by folate bearing 89Zr-Folate-FH (89Zr-11) than the non-folate bearing control, 89Zr-FH. In addition, folate (10) at 1 μM blocked more than 90% of the uptake of 89Zr-Folate-FH (89Zr-11, Fig 5e). 89Zr-Folate-FH (89Zr-11) uptake was temperature dependent, indicating uptake was by an energy requiring process like endocytosis (Fig 5f). On the other hand, uptake of 89Zr-FH was far lower and temperature independent. Folate groups on 89Zr-Folate-FH (89Zr-11) are bioactive, binding folate receptors on MDA-MB-231 cells.
89Zr-Cy5.5-Protamine-FH (89Zr-16) bioactivity.
To examine the membrane translocating bioactivity of the arginine-rich protamine peptide on the NP surface, we incubated 89Zr-Cy5.5-Protamine-FH (89Zr-16) with MDA-MB-231 cells at three concentrations (Fig 5g). This translocating bioactivity was greatly reduced at (4°C, Fig 5h). Protamine on 89Zr-Cy5.5-Protamine-FH (89Zr-16) is bioactive, inducing an uptake of 89Zr-Cy5.5-Protamine-FH (89Zr-16) (Fig 5h) than that of 89Zr-FH shown in (Fig 5f).
Our scheme of Fig 1 provides fluorescent and/or 89Zr labeled NPs (Cy5.5-Azide-FH (8), 89Zr- Cy5.5-Azizde-FH (89Zr-8), Cy5.5-Alkyne-FH (9) and 89Zr-Cy5.5-Alkyne-FH (89Zr-9)) that are internalized by the phagocytic monocyte/macrophage system, an internalization that can occur either in vivo or ex-vivo . Scheme 1 also provides a chemistry for the in vitro loading of non-phagocytic cells such as T lymphocytes, which do not internalize magnetic nanoparticles unless a translocation signal is involved [35, 37, 41]. Specifically, protamine can be attached to the FH NP that is internalized by mesenchymal stem cells and U87 cells . Thus some NPs described here will be internalized by the monocyte arm of the immune system while others (e.g. the protamine constructs 89Zr-Cy5.5-Protamine-FH (89Zr-16) or Cy5.5-Protamine-FH (16)) will be internalized by its lymphocyte arm. The NPs described here can be used for the ex-vivo labeling, and subsequent tracking of the various arms of the immune system.
We have shown that both azides and alkynes can be attached to FH and withstand HIR, allowing a wide range of click chemistry surface functionalizations with radioactive or non-radioactive NPs. However, with iron oxide NPs like FH the reducing conditions used for some click reactions can reduce ferric to ferrous ions, a reaction evident by a change from a reddish to a black/brown color. Our conditions for copper mediated click reactions of the Alkyne-FH (5) with Azide-Cy5.5 (7) or Azide-Folate (10) were 0.2 mg Fe, 15 min, room temperature with molar ratios of 67 / 1 / 1 / 37 for Fe / Cu / (BimC4A)3 / Na.Ascorbate in DPBS. In addition the copper catalyst should be freshly made and solutions should be oxygen free (see S1 File). As shown in Table 1, the measurements of nanoparticle size and relaxivity indicate a lack dissolution or aggregation due to surface functionalization. Copperless click reactions between Azide-FH (4) and click reactive, strained cyclic DBCO were used with RGD and protamine peptides; these are free from the issues than arise with the use of reductive conditions.
To determine the numbers of chemically reactive azide or chemically reactive alkyne functional groups on the NP surface we employed the “Before and After Fluorochrome Reaction and Subtraction Method.” This was done because of the shortcomings of other analytical methods discussed below, with Cy5.5 selected as an optical reporter because of its high extinction coefficient, its lack of spectral overlap with iron oxides (see Fig 2b), and the commercial availability of click reactive Cy5.5s. Shortcomings of other analytical arise when engaging in defining nanoparticle chemistry for nanoparticles used in mouse imaging. This arises because typically small quantities of nanoparticles are synthesized and used, since one milligram is sufficient to inject four 25 g mice at a typical dose of 10 mg Fe/kg. Hence analytical methods for this setting must consume small amounts of NPs, as well as overcoming NP superparamagnetism (no NMR) and the intense UV and visible absorption of iron oxides. Here we show how reactions with a commercially available fluorochrome can be used to obtain the number of biomolecules per NP. By reacting commercially available, click reactive Cy5.5s with NPs, the number attached Cy5.5s can be determined spectrophotometrically, with the number of bioactive groups, being the difference between the number of Cy5.5s attached before and number attached after their reaction the biomolecule. (Fig 3). With the high extinction coefficient of Cy5.5, the reaction of Cy5.5 with NPs uses only about 0.02 mg Fe to interrogate the status of the NP surface. Conventional methods of analyzing NP surfaces, e.g. FTIR or SIMMS  require substantially greater NP amounts and specialized instrumentation. With this “Before and After Fluorochrome Reaction and Subtraction Method” the numbers of folates or RDGs were determined through the reaction of Cy5.5. The method is useful when the number of attached targeting groups per NP is high, and difference of the before after reaction (p = m- q, Fig 3) is large. However, to determine low numbers protamines attached per NP, (a low number being necessitated by protamine induced NP agglomeration, see below), we synthesized a DBCO-protamine-Cy5.5 (15, Fig 4, see S2 File), and determined the numbers of protamines per NP directly.
With regard to steric blockage with Cy5.5 dye attachment to the Feraheme nanoparticle surface, steric blockage is not seen because of the Feraheme nanoparticle’s extremely large dimensions compared to a Cy5.5. The Feraheme nanoparticle has diameter of 25 nm, equivalent to a globular protein of 750 kDa . The Cy5.5 dyes have molecular weights of about 700–800 daltons, so that attachment of, for example 20 Cy5.5s (MW = 16,000 Da), is readily accommodated on the Feraheme nanoparticle. Thus we have not observed steric blocking and the number of Cy5.5s can be used to represent the reactive sites under click chemistry surface modification employed in this study.
A consideration when performing NP surface chemistry on FH NP is avoiding NP agglomeration. FH (1) has a strong negative charge over a wide pH range, with a Zeta potential at pH 7.36 of -30.55 mV . With our chemistry, FH (1) tolerated the attachment of negatively charged Cy5.5, the cRGD peptide or folate at levels of 10–20 targeting groups per NP with a maintenance of NP size of less than 50 nm, see Table 1. However, when highly positively charged protamine was employed, agglomeration was initially obtained. Protamine 1 consists of 32 amino acids of 22 of which are positive arginines and the rest are neutral amino acids . (Protamine is a mixture of 4 similar peptides.) To attach the smaller numbers of protamines consistent with NP stability, and determine the number of protamines per NP, we synthesized a protamine with N-terminal DBCO and C-terminal Cy5.5 (DBCO-protamine-Cy5.5 (15), see S2 File), and determined the number of protamines per NP directly from the attachment of DBCO-Protamine-Cy5.5 (15), see Fig 4.
Our surface modifications with Cy5.5 fluorochromes, folate, RGD peptide and protamine employed materials with molecular weights of less than 5000 daltons. Separation of radioactive nanoparticles from these low molecular weight, surface functionalizing compounds is readily accomplished with PD-10 and Sephadex G-50 columns. With our method, a wide range of nanoparticle surface functionalization strategies can be employed with similarly sized materials. However, use of larger biomolecules like antibodies will required a different separation method and may occur far more slowly under the conditions used here.
The combination of heat-induced NP radiolabeling and click chemistry NP surface functionalization outlined in Fig 1 provides a number of positive features. First, it employs Feraheme NPs as a starting NP, which is an approved drug. FH NPs have well-described physical properties,[9, 44, 45] a well-understood metabolism,[46–48] low toxicity,[49, 50] low batch to batch variability, and the sterility required of a parenteral pharmaceutical. Due to the superparamagnetism of its iron oxide core, FH (1) has been widely used as an MR contrast agent, which further aids in understanding diagnostic applications of this NP. Second, the carboxyl groups of FH’s carboxymethyldextran (CMD) coating endow the NP with extraordinary heat stability[11, 12, 52] and can be reacted with primary amines.[13, 42] CMD’s carboxyl groups are not involved in the radiocation binding of the HIR reaction based on electron magnetic resonance indicating their binding to the iron oxide surface and the ability to perform HIR with NP’s lacking carboxyl groups.  Thus, we have modified some of these carboxyl groups to obtain alkyne and azide functionalized NPs as shown in Fig 2. Third, FH’s heat stability allows heat-induced radiolabeling, which permits the bonding of three cations used in nuclear imaging (111In, 89Zr, 64Cu) without altering the NPs physical properties or pharmacokinetics.[4, 5] Here we demonstrate a fourth advantage of FH NPs, that alkyne and azide functionalized FH NPs (Azide-FH, 4 & Alkyne-FH, 5) survive the HIR reaction (yielding 89Zr-Azide-FH, 89Zr-4 & 89Zr-Alkyne-FH, 89Zr-5). This allows syntheses of nonradioactive Azide-FH (4) and Alkyne-FH (5) at modest pace and desired scale, followed by HIR, followed by the rapid click chemistry attachment of biomolecules under mild reaction conditions (Fig 3). Here Azide-FH (4) or Alkyne-FH (5) were radiolabeled, surface modified, and purified in total time of less than 4 h, which is short compared to the radiochemical half-lives of 64Cu, 111In or 89Zr.
As summarized in Table 1, the HIR/click combination is a powerful approach for generating multifunctional nanomaterials, which allows detection of the NPs made here by MRI (FH’s superparamagnetism), by fluorescence (click attachment of fluorochromes), and by radioactivity (HIR). In the future, the general strategy outlined in Fig 1 may be used to obtain multimodal NPs for labeling circulating monocytes or resident macrophages, for targeting NPs to cells expressing receptors (e.g. folate receptor, RGD binding integrins) or for the ex-vivo labeling cells through the membrane translocating activity of protamine.[35, 37, 41] The reactions demonstrated here, use of the widely available and well understood FH (1) for the synthesis of the nonradioactive Alkyne-FHs (5) and Azide-FH (4) intermediates, followed by HIR, then by click chemistry modification of the NP surface biomolecules, provides a path to the design of multimodal/multifunctional NPs suitable for a wide range of diagnostic and therapeutic applications.
S1 Fig. RCP analysis for 89Zr-Azide-FH (89Zr-4).
S2 Fig. RCP analysis of 89Zr-Alkyne-FH (89Zr-5).
S3 Fig. RCP analysis 89Zr-Cy5.5-Azide-FH (89Zr-8).
S4 Fig. RCP analysis 89Zr-Cy5.5-Alkyne-FH (89Zr-9).
S5 Fig. RCP analysis 89Zr-Folate-FH (89Zr-11).
S6 Fig. RCP analysis 89Zr-Cy5.5-Folate-FH (89Zr-12).
S7 Fig. RCP analysis 89Zr-Cy5.5-Protamine-FH (89Zr-16).
S8 Fig. Structures of RGD-DBCO, Folate-azide, Cy5.5-DBCO, Cy5.5-azide.
S1 File. Preparation of the stock solution for copper click catalyst.
- Conceptualization: LJ HY.
- Data curation: LJ HY MW GEF MN CK.
- Formal analysis: HY CK MW.
- Funding acquisition: LJ GEF MW CK MN.
- Investigation: HY MW CK.
- Methodology: LJ HY.
- Project administration: LJ HY GEF.
- Resources: HY MW CK.
- Supervision: LJ HY GEF MN.
- Visualization: LJ HY.
- Writing – original draft: LJ HY MW GEF MN CK.
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domain: chemistry


text:
The Capitals brings you the latest news from across Europe, through on-the-ground reporting by EURACTIV’s media network. You can subscribe to the newsletter here.
Before you start reading today’s edition of the Capitals, feel free to have a look at the video “Germany’s ‘crisis’ EU Presidency explained” by Claire Stam.
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In today’s news from the Capitals:
Italy’s ruling parties are still bickering over whether to make use of the European Stability Mechanism (ESM) to recover from the COVID-19 crisis. Money, both grants and loans, from the much-awaited Recovery Fund is expected in the second semester of 2021 and until then, EU member states can only use loans from several already agreed tools, such as ESM.
However, these loans will add to the countries’ public debt, which causes fear in many EU member states. The European Commission’s projection for Italy’s debt is at 159% of GDP.
Moreover, ESM loans come with conditionality attached and at the political level, the word bailout is rife with negative connotations and “banished”, especially in Europe’s south.
Nicola Zingaretti, leader of the Democratic Party, has urged coalition partners from the Five Star Movement (M5S) to stop wasting time and accept the ESM as it can provide “resources we have never seen before. EURACTIV Italy’s Alessandro Follis takes a closer look.
Plans to increase the minimum wage. Yesterday (30 June), Germany’s independent minimum wage commission laid out its plans to raise the minimum wage in the coming years from the current €9.35/hour to €9.50/hour from 1 January 2021 and to €10.45 from 1 July 2022. To put these changes into effect, the German government will have to issue new regulations which are generally based upon the commission’s recommendations.
While many welcomed the increases, some, such as the Social Democrats (SPD), said that €10.45/hour by 2022 does not go far enough. “Good that [the minimum wage] is to be increased now […] It is also clear that this is still too little! […] We are fighting for a minimum wage of at least 12€!” tweeted SPD General Secretary Lars Klingbeil. (Sarah Lawton | EURACTIV.de)
Meanwhile, Germany wants stronger AI regulations on the EU level, according to a statement released yesterday. Philipp Grüll looks into it.
Macron reshuffles cabinet as PM gets elected. After the defeat of French President Emmanuel Macron’s party at the municipal elections on Tuesday (30 June), the president will have a new government before 8 July, France Info reported.
That is because the current prime minister, Édouard Philippe, will be heading back to Le Havre (Normandy), where he has been re-elected last weekend. To fill in his spot, Macron is expecting proposals from the presidents of the Senate, the Assemblée Nationale and the Economic, Social and Environmental Council. The Greens, however, have already refused to be part of Macron’s new cabinet despite making significant gains in the municipal elections.(Anne Damiani| EURACTIV.fr)
New tool to test event health safety. As a ministerial decree approving the next phase of relaxation of COVID-19 lockdown measures from 1 July has been published, all event organisers in Flanders, Brussels and Wallonia will be able to test their event for possible health risks online via the COVID Event Risk Model (CERM), the so-called “event matrix”. The CERM was developed to facilitate the restart of the event sector and is applicable to all sporting, cultural, tourist or business events. The model was approved by GEES (the pandemic expert group that advises the government) and is based on 20 parameters to measure the safety risk. (Alexandra Brzozowski, EURACTIV.com)
Defence minister defends reform plans. After Defence Minister Klaudia Tanner’s new vision for the Austrian military (ÖVP) was met with a wave of protests last week, she defended her plan and insisted on the need for reform on Tuesday (30 June) but admitted her way of communicating it could be “debated”.
Since last week’s announcement, Austrian President Alexander van der Bellen has requested an ad-hoc discussion and many others have criticised the minister’s plan for taking away conventional defence activities from the military and investing them with more tasks related to cybersecurity, anti-terrorism and disaster management, which, according to her, pose more realistic threats. (Philipp Grüll | EURACTIV.de)
Minority government showing cracks. Partners in the Swedish minority government, often referred to as a “forced marriage” of the Social Democrats (SDP) and the Green Party, are not seeing eye to eye on the issue of migration. The two usually rely on support from the two opposition parties – the Center Party and the Liberals – to block the Sweden Democrats (SD).
Prime Minister Stefan Löfven’s SDP is pushing for parliament to adopt new legislation that would define and tighten immigration limits, which the Greens strongly oppose. The SDP is, therefore, relying on the main opposition party, the liberal-conservative Moderate Party, to vote in favour, thus side-tracking its coalition partner, the Greens. While it remains to be seen how this will affect the minority government in the long-term, one thing is for certain: No one wants new elections. (Pekka Vänttinen | EURACTIV.com)
Government will have to nationalise TAP, says minister. The Portuguese government said it will have to nationalise national flag carrier TAP after the state’s proposal for a loan of up to €1.2 billion with conditions had been rejected on Monday (29 June) by the company’s board of directors.
The proposed contract for the loan will still be submitted to the private partner, Atlantic Gateway, whose representatives abstained from voting on the board of directors.
“If the partner does not accept the conditions of the Portuguese state, we will have to intervene in the company, nationalise the company, yes,” said Infrastructure Minister Pedro Nuno Santos. The Portuguese airline posted losses of €395 million in this year’s first quarter as it was heavily impacted by the COVID-19 pandemic. (Maria João Pereira and Diogo Caldas, Lusa.pt)
Also read: Coronavirus prompts airline nationalisation spree
COVID-19 crisis heavily impacts GDP. The Spanish economy slumped 5.2% in this year’s first quarter compared to the previous one as a result of the coronavirus crisis, EURACTIV’s partner EFE reported. This marks an unprecedented decline of the local economy, Spain’s National Statistics Institute (INE) reported.
The GDP’s decrease is even bigger than the impact of the 2008-2009 crisis as investments fell by 5.7% since last March as the economy went into “hibernation” during the state of alarm from 14 March to 21 June. EURACTIV’s partner EFE reports.
Information on travel to Greece as of 1 July. As the summer season officially launches today in Greece, the government has presented detailed information about those who wish to travel to Greece this summer. See all the details here.
Also read: EU lifts travel restrictions for 15 third countries from 1 July, excluding the US
Elections conducted in professional manner. The first round of Poland’s presidential elections, which took place on Sunday (28 June) and sent President Andrzej Duda (43.5%) and Warsaw mayor Rafał Trzaskowski (30.46%) into the run-off, was conducted in a professional manner despite the lack of a clear legal situation. This is according to the Office for Democratic Institutions and Human Rights (ODIHR) of the Organisation for Security and Cooperation in Europe (OSCE).
ODIHR noted, however, that “the public broadcaster became a campaign tool for the incumbent president, and some media messages were clearly xenophobic and anti-Semitic”. It also noted that “the campaign itself was characterised by negative rhetoric on the part of the leading candidates, which further exacerbated the already confrontational atmosphere”. (Łukasz Gadzała | EURACTIV.pl)
Sassoli responds to Czech government criticism. “I wish to take the opportunity to recall that the EU Treaties give the European Parliament the power and the duty to give or deny the discharge to the European Commission on the implementation of the EU budget, in the exercise of its powers as a budgetary authority and of its function of democratic oversight,” European Parliament President David Sassoli wrote to Czech Prime Minister Andrej Babiš. The official letter was sent following a wave of criticism coming from the Czech government after the Parliament voted in favour of a resolution slamming the Czech PM over alleged conflicts of interest related to EU subsidies received by his former business empire.
“The resolution adopted by the European Parliament is, therefore, to be considered as part of this process and as an act stemming from its powers of budgetary control and democratic oversight, that should be regarded by all EU actors with the utmost respect,” the European Parliament chief added.
In other news, the Czech Republic decided to reduce the EU’s list of 15 non-EU states from where travel to Europe will be possible starting today (1 July). Czech borders only opened for Serbia, Montenegro, Thailand, Canada, Australia, New Zealand, Japan and South Korea, meaning that Algeria, Georgia, Morocco, Rwanda, Tunisia and Uruguay – which all feature on the EU’s list – have been left out.
“We want to motivate the countries to start treating us the same, to enable our citizens to travel there without conditions. This is why foreigners from these countries are not able to travel to the Czech Republic for the time being. But we are prepared to react very flexibly to a changing situation. This is the introduction of reciprocity,” Foreign Minister Tomáš Petříček said. (Aneta Zachová | EURACTIV.cz)
‘Black Lives Matter’ slogan is racist. Czech President Milos Zeman has described the “Black Lives Matter” slogan as racist. “I say that the slogan ‘Black Lives Matter’ is a racist slogan because all lives matter,” AFP quoted Zeman as saying on Tuesday at the US embassy in Prague.
Ruling party proposes to retroactively reevaluate all academic theses. As a solution to the plagiarism of politicians, all academic theses would have to undergo a control test, whose result would apply retroactively, according to a proposal made by Prime Minister Igor Matovič’s ruling OL’aNO party. Those who do not pass the test would lose their academic degrees. “Should this mean people would lose their jobs, so be it,” the PM added.
This comes after a scandal involving parliament Speaker Boris Kollár (We are family), who, despite media reports pointing out that he had largely plagiarised his master’s thesis, would not be affected by such a reform because his position does not require a university education. However, Education Minister Branislav Gröhling (Freedom and Solidarity) has already said this proposal is practically impossible. (Zuzana Gabrižová | EURACTIV.sk)
NEWS FROM THE BALKANS
Europol should intervene in Bulgarian PM threats against MEP. EU socialists chief Iratxe Garcia has sent a letter to EU Parliament President David Sassoli calling for the intervention of Europol in alleged “threats” by Bulgarian Prime Minister Boyko Borissov against a member of the EU House, Elena Yoncheva.
“Such a reaction from the Bulgarian authorities is highly regretful. Especially when taking into account that Bulgaria is, according to Transparency International, the country with the highest level of corruption in the European Union, and Ms Yoncheva, also a former journalist, has for a long time been actively engaged in the fight against it, in particular in a campaign to expose corruption at the highest government levels,” Garcia said. (Sarantis Michalopoulos)
See the letter here.
Romania’s major goals in budget negotiations. Romania’s main objective in the EU budget negotiations is to obtain the highest possible allocations for the cohesion and farming policies, President Klaus Iohannis told European Council chief Charles Michel on Tuesday.
Romania wants the traditional policies of the EU to continue to play a big role in the EU’s future budget, given that they are the most important, well-oiled instruments to finance investments in the EU, and they can be very efficient in crisis situations, according to a news release of the presidency, published following a videoconference between the two leaders after the EU Summit of 18-19 June. EURACTIV Romania’s Bogdan Neagu reports from Bucharest.
In other news, Romania’s parliament selected a new seven-member board for the country’s agency in charge of applying the Romanian and EU anti-discrimination legislation – the National Council for Combating Discrimination, or CNCD.
The board consists of some returning members, including current president Asztalos Csaba, but also new faces, such as an MP who notably made a grammar error in his candidacy papers and a former police officer who had been charged with car theft. A total of 26 candidates ran for the seven positions, but two of them – including former MEP Doru Frunzulica (S&D) – withdrew their candidacies before the parliament vote. (Bogdan Neagu | EURACTIV.ro)
TV clash ahead of parliamentary elections. Ahead of parliamentary elections due on 5 July, HDZ president and Prime Minister, Andrej Plenković, and SDP president and Restart Coalition leader Davor Bernardić clashed in a TV debate broadcast on RTL television on Monday evening (29 June).
While the two candidates opposed the formation of a grand coalition and claimed their first move would consist of adopting a law for the post-earthquake reconstruction of Zagreb, they clashed on minimum wage issues, the country’s COVID-19 response and European policies. Plenković even accused Bernardić of lacking knowledge about European topics. Read more about the debate here. (Karla Juničić, EURACTIV.hr)
Interior minister quits over police raid. Slovenia’s interior minister Aleš Hojs and the country’s police director Anton Travner resigned on Tuesday (30 June) because of surprise police raids on houses of several government officials, investigating the purchase of medical ventilators. EURACTIV’s Zoran Radosavljević has the full story.
[Edited by Sarantis Michalopoulos, Daniel Eck, Zoran Radosavljević]
Source link Google News


text:
Three college students on a road trip across the Southwest experience a detour – the tracking of a computer genius who has already hacked into MIT and exposed security faults. When the trio find themselves drawn to an eerily isolated area, suddenly everything goes dark. When one of the students regains consciousness, he finds himself in a waking nightmare.
|Release Date||:||March 15, 2014|
|Genres||:||Thriller, Science Fiction|
|Production Co.||:||Signal Film Group, Low Spark Films, Automatik Entertainment|
|Production Countries||:||United States of America|
|Director||:||William Eubank, Darryl Garcia Jr.|
|Writers||:||Carlyle Eurbank, William Eubank, David Frigerio|
|Casts||:||Brenton Thwaites, Olivia Cooke, Beau Knapp, Laurence Fishburne, Robert Longstreet, Lin Shaye, Jeffrey Grover, Patrick Davidson, Roy Kenny, Timothy Holmes, Ricardo Campos, Drew Sykes|
R U Agitated?
Original Title : Batman Release Date : June 23, 1989 Popularity : 16.488 Original Title : Green Lantern: Emerald Knights Release Date : April 9, 2011 Popularity : 8.329 Original Title : Batman Returns Release Date : June 19, 1992 Popularity : 16.182 Original Title : Shazam! Release Date : March 23, 2019 Popularity : 41.238 Original Title : Justice League Release Date : November 15, 2017 Popularity : 43.313 Original Title : The Dark Knight Release Date : July 16, 2008 Popularity : 35.402


text: The general field of molecular simulation provides a wide spectrum of methods for studying the structure and function of biomolecules. Depending on the scale and question of interest, appropriate approaches may range from ab initio quantum mechanical calculations (when detailed aspects of and changes in electronic structure must be considered) to Brownian dynamics and coarse-grained molecular dynamics (to track large scale conformational motions, diffusion, and inter-molecular interactions). The entire range of molecular simulation methods has been fruitfully applied to a range of flavoenzymes, allowing researchers to address everything from the specific intermediates involved in the photoreactions of flavin chromophore-containing light sensors, to the very long timescale motions induced by covalent modifications to bound flavin. The unique challenge posed by flavoproteins to all types of molecular simulation arises from the chemistry of the flavin isoalloxazine moiety, which presents an unusually large delocalized electron system which must be carefully treated in order to represent its contributions to the overall behavior of the system. Here we outline the particular considerations required for appropriate treatment of flavoproteins in simulations ranging from electronic structure calculations to long-timescale modeling of flavoprotein conformational transitions.


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According to new research over 60 percent of people in Britain are unaware that gay and bisexual men can become foster parents.
The worrying statistic shows that there’s a long way to go in order to normalise the idea that LGBT+ people can become foster parents.
Across the UK, around 60 percent of people were unaware that LGBT+ people are eligible to foster children.
According to Five Rivers Child Care, there are over 900 children and young people who need foster homes across London yet there are common misconceptions around who is eligible to foster, from age through to living circumstances and sexual orientation.
Over 60% of people across London and the UK as a whole are unaware that same-sex couples or those from the LGBT+ community are eligible to foster. A misconception Five Rivers Child Care is keen to eliminate this February, which marks LGBT History Month, in a bid to recruit more foster carers from currently underrepresented communities.
In addition to serious misconceptions surrounding LGBTQ rights to foster, a further 60 percent of people across the UK believe that those in rented accommodation are unable to foster, and 40 percent of us believe that those without their own children wouldn’t be allowed to be foster carers.
Commenting on the findings, Martin Leitch, Head of Fostering Operations at Five Rivers Child Care, said, “The findings from our recent research paint a massively outdated picture of the fostering industry but it highlights that more needs to be done to reach those within LGBTQ communities to tell them about the career opportunities available to them in fostering. And it’s not just a career, it’s a life choice. It’s deciding to change the life of a vulnerable young person that otherwise might not have a home or a family. It’s extremely important to have a diverse range of foster carers across the country to reflect the diverse nature of the children and young people entering the system and provide role models from all walks of life. We already have some fantastic carers from the LGBTQ community who are enriching children’s and young people’s lives on a daily basis.
“We want to make it common knowledge that anyone can foster, providing they have a spare room and they are over the age of 21. A foster carer is not determined by whether they own a house, or whether they’re married or have children. Single people, those in rented accommodation, those with disabilities and those from the LGBTQ community all have the opportunity to make great foster carers.”

domain: sociology


text: One of the mistakes many of us make is that we feel sorry for ourselves, or for others, thinking that life should be fair, or that someday it will be. It's not and it won't. What gives us the right to expect to be rewarded for hard work or the good things we do? We must do what's right for our own sake. The nice thing about surrendering to the fact that life isn't fair is that it keeps us from feeling sorry for ourselves. Anyway, it's not life's job to make everything perfect, it's our own challenge.

domain: literature


text:
The key difference between source and sink in plants is that source in plants is the site of food production using biological processes while sink in plants is the site of food storage for later use.
Source and sink in plants are two important terminologies used in phloem translocation. Phloem translocation is the process of transportation of the produced food (sucrose) in plants. Thus, sources in plants are the sites that are capable of producing sucrose; plant leaves are the main sources in plants. Whereas, the sites such as stems and roots which store the produced food are the sinks of the plant.
1. Overview and Key Difference
2. What is Source in Plants
3. What is Sink in Plants
4. Similarities Between Source and Sink in Plants
5. Side by Side Comparison – Source vs Sink in Plants in Tabular Form
What is Source in Plants?
Source of the plant is the site where photosynthesis primarily takes place to produce plant food in the form of sucrose. These sites mainly include the plant leaves. Plant leaves produce sucrose using carbon dioxide and water as their raw materials. However, this process takes place in the presence of sunlight. Chlorophyll pigments trap light energy from the sunlight to produce sucrose. Sucrose is the main form of sugar which can be transported in the phloem tubes. Thus, transportation of sucrose from the production site to its target is called phloem translocation. Phloem loading, which is the process of loading the sucrose to the phloem, takes place at the source.
In addition, the source of a plant can also be a site where nutrient entry takes place; for example, root hairs. The root hairs act as a source for nutrient uptake. Nutrients such as nitrates, nitrites, and phosphates are taken up by plant root hairs. Therefore, they are also source points in plants.
What is Sink in Plants?
Sink in plants is the site where the storage of produced food takes place. Thus, the transportation of produced food in the source will end up in the sink. Therefore, the end point of phloem translocation is the sink. The phloem will unload its contents at the point of the sink. Hence, phloem unloading takes place at the sink. The main sites of the plants which act as a sink are roots, stems, and flowers. The sink stores the produced food as starch. Therefore, the iodine test for starch identification can be used to determine the presence of starch in the sink sites.
In addition, during the amino acid metabolism in plants, the storage of amino acids takes place in root tips. Therefore, root tips also can act as a sink for plants to store amino acids.
What are the Similarities Between Source and Sink in Plants?
- Both source and sink are important in phloem translocation of sucrose.
- Both are present in mature plants which are vascular plants.
- However, non-vascular plants lack both source and sink.
- Moreover, transportation between the source and sink depends on the osmotic pressure.
- And, it takes place by mass flow.
What is the Difference Between Source and Sink in Plants?
In terms of phloem transport, the source and sink play major roles. The source produces the food required for translocation, whereas the sink stores the food brought by translocation. So, this is the key difference between source and sink in plants. Thus, photosynthesis rapidly takes place in the source while photosynthesis does not take place in the sink. Therefore, we can consider this too as a difference between source and sink in plants.
However, the sink is important in storing the produced sucrose, which can be later utilized for the activities of the plant while source does not involve in food storage. Moreover, a major difference between source and sink in plants is that the source loads sucrose for translocation while sink unloads the loaded sucrose.
The below info-graphic presents more information regarding the difference between source and sink in plants.
Summary – Source vs Sink in Plants
Source and sink are important concepts in phloem translocation. Source refers to the site where plants produce their food using photosynthesis. In contrast, sink refers to the site where the plant stores the produced food. Therefore, this is the key difference between source and sink in plants. Furthermore, loading of sucrose to the phloem takes place at the source, whereas unloading of the food takes place at the sink. The typical example of a source site is the plant leaf. Meanwhile, plant roots, stems and flowers are several sinks of a plant.
1. “Source and Sink in Phloem Translocation | Plant Physiology.” Biology Discussion, 12 Dec. 2016, Available here.
2. “Transport of Materials from Sources to Sinks at Different Seasons.” Biology Notes for IGCSE 2014, Available here.

domain: agronomy