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February 2014 Archive

Posted by Chris on February 7th, 2014  ⟩  0 comments

Apparently, I am a dog person. It might actually be truer to say that I like all animals, but I’m sure that if you were able to ask my dog her opinion, she would tell you: I am a dog person. As a product of my culture, I spend an inordinate amount of money and time on my dog. Studies show that Americans spent over $60 Billion on their pets in 2013, with slightly more than half of that on just their dogs alone. Specifically, nearly 46 million households have provided a comfortable, all-inclusive, resort-like home for nearly 80 million dogs in the United States in 2013. Those trends have been steadily increasing for years and do not look to taper off anytime soon.

As we improve the quality of life for our dogs, their life expectancy has increased, just like ours did in the throughout the 20th century. It’s no surprise, then, that dogs are seeing an increase in cancer rates, partially due to their extended life expectancies…again, similar to our own history of complications with increased life expectancy. One of the most common forms of bone cancer in dogs is called Osteosarcoma or OSA. Its prevalence most often strikes in larger, and older, dogs and accounts for about 85% of canine skeletal tumors. OSA CancerOSA occurs often around the leg joints and treatment usually involves amputation followed by chemotherapy. However, even with treatment, the prognosis is very poor. Approximately 90% of dogs succumb to metastasis and the average life expectancy, post treatment, is only 6 months. Tragically, there is just a 50% chance of surviving a year and but a 25% chance of our best friends surviving beyond that point.

As with all cancers, the key to survival is earlier detection and earlier treatment/therapy. But it’s not as if our beloved pets can tell us when something hurts or just doesn't feel right. More often than not, they play through the pain, ignoring all but the worst of their injuries and ailments. And let’s face it, most of us pay less attention to the small clues they do give us than we should, further complicating the job of our veterinarians. To help address this tragic problem, a group from the College of Veterinary Medicine in Oregon, began the introductory process of identifying and defining the surface-exposed proteins (SEP) of OSA cancerous cells.

Milan Milovancev et al. used a biotinylation/streptavidin enrichment process to label and identify with Mass Spectrometry more than 100 putative SEP candidates from verified OSA cell lines as compared to normal osteoblasts. Utilizing the strength of the biotin-streptavidin association, Milovancev’s group biotinylated the surface-exposed proteins in the cells (for a similar system, see Gold Bio’s Biotin conjugation kits), lysed the cells to release the proteins and separated the biotinylated proteins using streptavidin-labeled beads…simple, clean and efficient.

Of course the enrichment process, by its own method, skews the quantification of the protein levels in the tumor cells. But the purpose of this study was only to find putative candidates, which can then be further studied in depth at a later time. To that end, Milovancev’s group set out to validate these initial results with secondary identification methods, such as RT-PCR, Western Blots and Immunocytochemistry. The follow up results mostly (but not completely) verified the results of the enrichment/MS identification method, meaning that while the system will do what it’s supposed to in general, Milovancev is just going to work harder to achieve a future, better refinement of the procedure.

As a dog person, I (and my dog) wish him well.


Milovancev, M., Hilgart-Martiszus, I., McNamara, M. J., Goodall, C. P., Seguin, B., Bracha, S., & Wickramasekara, S. I. (2013). Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts. BMC Veterinary Research, 9, 116.

Category Code: 79101 88231

Posted by Chris on February 13th, 2014  ⟩  0 comments

In the comfortable culture of the U.S., which we take for granted all too often, it can be difficult to remember the human plagues and diseases that have followed mankind throughout the millennia. It’s difficult to remember that we, ourselves, are the preferred breeding ground for a host of bugs, worms and parasites; not just idle or incidental carriers of these microscopic beasts, but their main course…dependent on our unique physiology in order to reproduce and survive.

Some of these diseases are famous, due to their pugnacious nature or severity of effect; diseases such as malaria, botulism, or River Blindness. Others are less well known, either due to their more limited environment or because their incidence in more developed countries is less common, such as the protozoan, Entamoeba histolytica. However, less common does not necessarily mean less destructive. Entamoeba is a group of anaerobic parasites that specifically target humans and primates. Typically passed from feces and water to another host in a cystic form, these protozoans mature in our digestive tracts and cause a disease called Amebiasis. Amebiasis is typically characterized by abdominal pain, amebic dysentery, bloody diarrhea, and fevers. The protozoans can also occasionally escape the colon and end up on other organs to cause liver, brain or lung abscesses. Current estimates are that nearly 50 million people, worldwide, suffer infection from E. histolytica, which result in around 100,000 deaths. However, only 10-20% of all infections become symptomatic, so the number of infections may actually be much higher. The rate of infection of E. histolytica in tropical countries in Central or South America, Africa and Asia is actually nearer to 50%! And when the numbers of E. histolytica are combined with similar, but non-symptomatic protozoans (such as E. dispar and E. moshkovskii), the total world count may be closer to 10% or 500 million infective cases.

Protozoan immunofluroescentThe most likely place to start looking for better methods of disease prevention of Amebiasis are the surface protein interactions between the protozoan and our intestinal walls which cause adherence and activation of the protozoan to its trophozoite stage. Cataloguing and defining these surface proteins is about as easy as identifying all of the various countries on Earth from a telescope on Mars. To date, only a smattering of about 20 of these surface proteins had been identified. Iris Bruchhaus and her group from the Nocht Institute for Tropical Disease wanted to blow that number away and settle once and for all what proteins need to be studied.

Bruchhaus’ group used a non-permeable biotinylation process (similar to Gold Bio’s Biotinylation kits found here) to bind with all of the surface proteins on the HM1:IMSS strain of E. histolytica. Those biotin conjugated proteins were then easily isolated using a streptavidin agarose resin system (which you can also conveniently find here), and analyzed in NanoLC-MS/MS in order to identify the proteins. They found close to 700 proteins! Oddly though, nearly 50% of the proteins found they found did not show any specific membrane association. Bruchhaus’ group was able to further detail many of these isolated oddball proteins, showing that roughly 85% of them did have some cell surface interaction, even if not in the traditional sense. But this actually implies that the plasma membranes (and their surface associations) of these protozoans are not static and easily defined at all, but are a very dynamic, complex and interconnected weaving of molecules in constant exchange on and across the membrane.

That doesn’t necessarily make this type of work any easier. But with a large number these proteins identified, work can at least begin on some of the most important proteins, further characterizing the association with their human host cells. Who knows, maybe it’s actually one of these oddball proteins that only sometimes associates with the plasma membrane that account for the low percentage of the protozoan becoming symptomatic. Time will tell…


Biller, L., Matthiesen, J., Kühne, V., Lotter, H., Handal, G., Nozaki, T., Saito-Nakano, Y., Schumann, M., Roeder, T., Roeder, E., Krause, E., & Bruchhaus, I. (2014). The Cell Surface Proteome of Entamoeba histolytica. Molecular & Cellular Proteomics, 13(1), 132-144.

Category Code: 79101 88231

Posted by Chris on February 20th, 2014  ⟩  0 comments

I believe that our current quality of life has been most affected by the discovery of vaccines, even more so than by our use of antibiotics. I also firmly believe that the benefits of those vaccines outweigh any and all side-effects, disadvantages and the scare-mongering tirades about inerudite, nonsense-driven “research”. I am personally, extremely ecstatic that I never had to worry about getting polio or mumps or rubella. Though the shots hurt like crazy at the time, I can now silently thank my parents for listening to the “advice” of knowledgeable doctors and scientists who promised them I could have a better chance of life than the one their parents lived with. t is advice that I have also chosen to listen to for my own children, rather than let them fall prey to the ravages of the most terrifying diseases and plagues that our species has dealt with so poorly throughout millennia…until only just these last hundred years or so.

Unlike antibiotics, vaccines work with our body’s natural defenses by helping to build an initial bulwark against the specific antigens of a disease that can then be repopulated at a later time in the event that the disease manifests. You can think of vaccines more like an underground, resistance force laying the groundwork for a potential invasion as compared to an antibiotic’s nuclear missile strike after the invasion already took place. That cooperative action also means that we don’t have to worry about things like developing immunities to vaccines, since they are not the main actors on the stage of combat, only the catalysts for better host immune response.

There are, of course, some inherent risks involved with vaccines. Attenuated microorganisms in some vaccines may go rogue and develop into the full-fledged disease they originated from. A person’s immune response may also go hyperactive against the vaccine dose, causing traumatic problems. In therapeutically vaccinated patients, there may not enough of an immune response left in the body (for any of a variety of reasons) so that vaccination does little to help sufficiently subdue the disease. These are not trivial matters, to say the least, and doctors and scientists are constantly working to create better and more efficacious vaccines or vaccination systems. Recently, a group from the University of Navarra in Spain, under Juan Jose Lasarte, may just have found a novel method to improve how we produce vaccines.

Lasarte’s team had already previously discovered that the fusion of the Extra Domain A (EDA) of fibronectin to an antigen leads to an increase in its association with TLR4-expressing dendritic cells, causing greater immunogenicity of the antigen and greater induction of immunity. But the fusion of EDA to every single, different antigen is time-consuming, problematic and prohibitive. But what if they could find a more universal approach to creating the fusion? Lasarte’s team focused on building a hybrid EDA-linked streptavidin recombinant protein that could be simply and easily linked to any biotinylated antigen or protein due to the high affinity streptavidin naturally has for biotin. They called their new protein the “EDAvidin”.

One of the most important aspects of the streptavidin-biotin interaction is the requirement of the tetramarization of the streptavidin protein. Through a series of tests and comparisons, Lasarte’s group could see that the new EDAvidin still formed into the tetramer orientation required for optimal biotin interaction. Even better, they biotinylated an ovalbumin (OVA) protein (using something very similar to one of Gold Bio’s Biotin Labeling Kits) and successfully pulled the OVA proteins out of solution using EVAvidin in an ELISA assay. The binding affinity wasn’t quite as good as the original streptavidin-biotin, reaching only to 10-14 as compared to the original’s Kd of 10-15, but that’s still really good! The EDAvidin also maintained the proimflammatory activity that they noted in their earlier work with pure EDA, producing an immune response in the T cells of mice similar to that of antigens linked only to EDA.

All in all, EDAvidin presents the best of both worlds: a method to produce better acting vaccines that can be ubiquitously used with any protein that can be biotinylated, which is pretty much any protein. Lasarte’s method may end up becoming the building block for an entire new system of vaccine therapy, leading to better cancer tumor control and immunoresponse. I think that it’s just another brilliant use of an already brilliant system.


Arribillaga, L., Durantez, M., Lozano, T., Rudilla, F., Rehberger, F., Casares, N., Villanueva, L., Martinez, M., Gorraiz, M., Borrás-Cuesta, F., Sarobe, P., Prieto, J. & Lasarte, J. J. (2013). A Fusion Protein between Streptavidin and the Endogenous TLR4 Ligand EDA Targets Biotinylated Antigens to Dendritic Cells and Induces T Cell Responses In Vivo. BioMed research international, 2013.

Category Code: 79101 88231


PS. For an awesome example of what vaccinating does for us as opposed to not vaccinating, you can check out Penn and Teller explaining it in their unique, condescending (and humorous) way.


Posted by Chris on February 27th, 2014  ⟩  0 comments

It is probably a safe bet that everyone reading this knows at least one person who has diabetes. Diabetes is such a common disease in our culture, we hardly blink anymore when we find out that someone has it, as opposed to more ominous diseases like cancer, cardio-vascular disease or AIDS. Diabetes is the common name for Diabetes mellitus, but it is also a generalized term that combines two very different diseases that produce similar symptoms and effects. When we hear the word “diabetes”, most immediately think of the Type 2 version, the overweight and unhealthy, dietary disease that affects nearly 10% of all Americans and more than 300 million people worldwide. That’s not surprising, since Type 2 diabetes (T2DM) makes up approximately 90% of all diabetes cases. But there’s a huge difference between Type 2 and Type 1 diabetes (T1DM).

Whereas T2DM can be largely traced to poor nutritional and lifestyle choices, along with a smattering of genetic predispositions, which collectively gang up against the body’s insulin and prevent it from doing its job, rather like the fairy tale Cinderella’s step-mother and sisters preventing her from going to the ball. However, T1DM is actually an autoimmune disease in which the body’s immune system goes haywire and begins targeting the insulin-producing beta cells in the pancreas. Imagine how bad that infamous story would have been if Cinderella’s had been killed by her wicked step-mother immediately after her father died instead.

T1DM isn’t just insulin resistance, it’s the loss of most or all of the insulin in the body, due to the destruction of the cells that produce it. The end-effect looks identical to T2DM, which is why they are both commonly called the same thing, but where Type 2 can be possibly be reversed with proper diet and better exercise, Type 1 is a lifelong condition that has little to no chance of ever going away. Also, to date, T1DM cannot be prevented. While it’s known what happens, we still do not know why; whether it’s a simply genetic susceptibility or some kind of external trigger or some combination of the two or even something else that’s yet to be discovered.

However, scientists have noticed patterns of autoantibodies that have been able to predict the onset of T1DM. Autoantibodies for islet cells (also called islet of Langerhans), the cells responsible for producing the beta cells which monitor sugar levels and release insulin, for insulin, for glutamic acid decarboxylase (GAD65), for protein tyrosine phosphatase (IA-2) and for zinc transporter 8 (ZnT8) all account for some degree of T1DM onset. The more, different autoantibodies found, the higher the likelihood of eventually developing T1DM. The presence of these autoantibodies is sometimes called “latent autoimmune diabetes” and there are some doctors who believe that if these autoantibodies can be detected and suppressed early enough, then full onset T1DM might be preventable.

Since insulin autoantibodies (IAA) are usually the first to appear, they are an enticing target for scientists and it should be an easy and simple ELISA experiment, but IAAs are additionally bothersome in that they refuse to bind to human insulin which has been bound to an ELISA plate. Several other methods, such as RIA (radioimmunoassay) and ECL (electrochemiluminescence), have therefore been developed. But RIA depends on radiolabeled antigens, which are always a pain to deal with, and ECL requires high cost equipment and training and there have been reports of poor result correlation between labs. But what if there was a way to make that ELISA work after all? Well, a group from France, led by Nathalie Morel, think they’ve found a way.

Bridge ELISA

Morel’s concept is called a Bridge-ELISA. Binding the IAA to a biotinylated insulin in one of its antigen-binding domain (for a great system that can do just that, check out our Gold Bio Biotin Labeling Kits) allowed for easy detection via a streptavidin-conjugated tracer, and the other of its antigen-binding domains was bound to a GC300 hapten to help bind to an ELISA plate coated with the anti-GC300 monoclonal antibody MC159. Ultimately, Morel’s group developed a process that is faster, simpler to use and more reliable than ECL, and was safer (since there is no radioactive components) than the traditional RIA method, although it was only about 80% as sensitive as RIA in detecting IAA’s in T1DM children. Because it utilizes the standard ELISA format, practically every protein lab in the world can do it and automate the process for large-scale, faster results. And when it comes to saving insulin producing beta cells and possibly preventing T1DM, every small step forward might help to save a life you know.


Kikkas I, Mallone R, Tubiana-Rufi N, Chevenne D, Carel JC, Creminon, C., Volland, H., Boitard, C. and Morel, N. (2013) A Simple and Fast Non-Radioactive Bridging Immunoassay for Insulin Autoantibodies. PLoS ONE 8(7): e69021. doi:10.1371/journal.pone.0069021

Category Code: 79101 88231