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August 2013 Archive

Posted by Chris on August 8th, 2013  ⟩  0 comments

It’s extremely well known that our antibiotics are failing us. Alexander Fleming noticed it from the very beginning. Improper use of antibacterial agents leads to improved bacterial resistance to the agent. Throughout the decades since Fleming’s discovery of penicillin, we have watched as diseases have slowly (or quickly, in some cases) developed resistance to one antibiotic after another. The speed with which they are developing resistance has nearly surpassed the rate with which we can develop new antibiotics. And our newest antibiotics are losing their efficacy faster than their predecessors. It’s a viscous cause and effect cycle which we seem destined to lose.

As I searched for interesting research on Cephalosporins this week, I was reintroduced to this scary fact from several recent articles discussing the plight of increased resistance throughout the world to some of our most potent antibitics. First, I found a letter from Peter Collignon and colleagues to the CDC journal, Emerging Infectious Diseases, about the over-use of third generation cephalosporins in poultry through Europe. Collignon cited an ever rising tide of resistant E. coli (G3CREC) across Europe and that G3CREC-related illnesses have already been associated with over 900 hospital bed-days needed to treat the resistant infections and even resulted in 21 deaths in the Netherlands! The strangest part is that most countries have a “prudent use” policy in place concerning medical delivery of many antibiotics. At the same time, however, farm and veterinary implementation of these policies are lagging far behind. With the high rate of gene similarity between the G3CREC in humans and poultry in samples taken throughout Europe, it’s easy to see why the practice of large scale inoculations is so dangerous.

In another part of the world, Bhasker Thakuria and Kingshuk Lahon continued the cry for better “prudent use” polices in developing nations like India. There, it is cost prohibitive to do a full biological screening of every infection or illness. Through the lack of antibiotic susceptibility resources and the lack of guidelines for more common diseases, there is also a prevalence in using state-of-the-art antibacterial agents, such as later generation cephalosporins, to treat every patient’s illness, regardless of their specific needs. Thakuria and Lahon issued a call to improve the empirical use of drugs like cephalosporins before they become completely obsolete in our war against disease.

Finally, there was an article written by Douglas Call and associates detailing the possibility that increased antibiotic resistance might be related not just to our over-use of the drugs, but the downstream destinations of those drugs. Call found that metabolites of third generation cephalosporins, such as ceftiofur, excreted from cattle can still persist and remain biologically active for up to 3 days at room temperature (or several weeks at 4°C). And since 70% of ceftiofur is excreted through urine within 24 hours of injection, there is a high rate of probability that it will affect bacteria in the surrounding soil. The eventual resistant bacteria reenters the cattle through their environment, resulting in further resistance down the chain.

Our irresponsible use of antibiotics have created a pothole in the road to healthier living. Our continued irresponsible use will only serve to widen and deepen the hole we’ve made, eventually leading to the complete ineffectuality of our “antibiotic age” and the resurgence of some of mankind’s greatest diseases. In truth, perhaps it is not so much that our antibiotics are failing us, but that we are failing our antibiotics.

 
 

Collignon, P., Aarestrup, F., Irwin, R., and McEwen, S. (2013) C. D. C. Human Deaths and Third-Generation Cephalosporin use in Poultry, Europe (letter). Emerging Infectious Diseases, 19 (8), 1339.

Thakuria, B., & Lahon, K. (2013). The Beta Lactam Antibiotics as an Empirical Therapy in a Developing Country: An Update on Their Current Status and Recommendations to Counter the Resistance against Them. Journal of Clinical and Diagnostic Research, 7(6), 1207-1214.

Call, D. R., Matthews, L., Subbiah, M., & Liu, J. (2013). Do antibiotic residues in soils play a role in amplification and transmission of antibiotic resistant bacteria in cattle populations?. Frontiers in microbiology, 4 (193).

Category Code: 79102

Posted by Chris on August 1st, 2013  ⟩  0 comments

One of the questions we receive from time to time at GoldBio is whether purity really matters when it comes to chemicals. We can understand that question. There is a huge variety of “grades” with different purities available for almost every chemical on the market. Additionally, the grades/purities from one supplier do not always match exactly with that of a second supplier, so we know that it can be very difficult to compare otherwise seemingly equivalent products. What really are the differences between common grades such as Ultra Pure, ACS, Biotechnology, Reagent or HPLC? And which one should you use?

In a small lab, the answer is typically, “use whatever is already available!” But if you are designing an experiment from scratch or want the experiment done right, you want to make sure that the reagents you’re using are the right ones for the job. There are differences between the grade types that might be important for specific applications. For instance, "Ultra Pure" is a grade that signifies that the chemical has a purity exceeding more common grades. "ACS" is a grade for chemicals that specifically conform to the requirements of the American Chemical Society (for that chemical). However, Biotechnology Grade or Tissue Culture Grade might have an equal level of chemical purity (98-99% in some cases) as the Ultra Pure or ACS, but they are further purified to remove more specific contaminants, such as nucleases or bacteria, which could cause havoc in specific experiments. In those situations, are you really sure you want to use that ACS grade chemical (that’s been on the shelf for 3+ years and opened several dozen times) for your tissue culture experiment that might take you the better part of 3 months to conclude? Trust me, it really is better if you get the appropriately graded chemical.

Of course, chemical purity is still important. For instance, Goldbio’s Luciferin is 99.7-99.8% pure, the highest purity luciferin available on the market!** Most other suppliers have a minimum purity of only 98% (and sometimes, as low as 95%)! Maybe that doesn’t sound like much, but let’s do the math anyway. If 1 gram of 98.0% luciferin is dissolved in 25 ml buffer (a fairly standard dilution for in vivo assays) there would be 0.02 g of any potential contaminants. Again, that doesn’t sound like much, but that actually equates to 0.8 g per liter. If our theoretical contaminant has a molecular weight of 1000 g/mole, then the contaminant would be at a concentration of 800µM! That’s more than high enough to inhibit some enzymes in a cell. 

Luciferin purity is principally quantified by HPLC, and any impurities which may be left in the chemical are typically labeled as “unknown impurities”. The sensitivity and reliability of HPLC make common impurities fairly easy to distinguish and separate, so any unknown impurities may give you reason to pause. Of course, these unknown impurities may or may not be detrimental to an experiment. But just like in the previous example, are you really sure you want to trust a reagent with a lot of unknown impurities? Can you really be sure that potential impurities in your luciferin will be benign and have no side-effects on your 6-12 month mouse or rat study?

At GoldBio, we understand that question. It is why we strive to provide reagents, like luciferin, that are the best quality for your experiments. It is why we test our reagents personally to guarantee their efficacy. And that is why you trust GoldBio to provide those reagents for your research. 

It's just good science.

We would love to hear from you, so if you have any questions about any of our products, you can email us at: techsupport@goldbio.com!
 

  

Category Code: 79105 79102 88253 79108 79107 79109


 

Posted by unknown on August 5th, 2013  ⟩  0 comments

As more customers continue to turn to Gold Bio to provide high quality and affordable chemicals, we continue to strive to provide reagents that meet our stringent quality standards that we can bring to our customers at low prices. For the past few months we have feverishly been adding new products to our portfolio. We’ve added staples such as Tris and Hepes. Additionally, we now provide many more protease inhibitors for those of you making up your own cocktails. With the best prices around, if we have it, you don’t have to look anywhere else. We know that our reputation relies on the success of your experiments. We are dedicated to providing the highest quality products available. They are tested rigorously to make sure they are ready for your experiments and we back them up with a guarantee of making things right if they are not. The source and quality of our reagents is important to us because the success of your experiments is important to you. As we continue to grow our portfolio, be sure to always check goldbio.com for any reagents you need.

Right now, however, I want to focus on the addition of over 140 new antibiotics that cover a range of activity against gram-negative and gram-positive, aerobic and anaerobic, some broad spectrum as well as highly specific agents, we want to provide the best versatility for all of your research needs in the fields of microbiology and epidemiology. Many of these new antibiotics have water soluble forms for easier handling and are specifically useful for in vitro research and diagnostic applications in biotech, pharmaceutical and academic laboratories.

Category Code: 88261

Posted by Chris on August 15th, 2013  ⟩  0 comments

Aminoglycosides are one of the largest and most common classes of antibiotics we use on a daily basis. The founding member of this group, Streptomycin, was discovered early in the golden age of antibiotics and was the first antibiotic found to be efficacious in treating tuberculosis. In the decades that followed, many new aminoglycosides were discovered or synthetically developed, including: kanamycin, spectinomycin, amikacin, apramycin and hygromycin. These drugs have been a bulwark of our antibiotic armament over the years due to their effectiveness against both gram positive and gram negative bacteria, their synergism with other antibiotic classes and their generally low cost. But as with all antibiotics, organisms have been developing resistance to them as fast as we have been able to develop new variants.

There are three main methods by which organisms have gained resistance to aminoglycosides: decreased intracellular concentration due to active efflux of the antibiotic (as with lung isolates of P. aeruginosa), targeted modifications of genes like rpsL or rrs which change their protein structure slightly, but significantly, and most commonly by enzymatic modification by aminoglycoside-modifying enzymes (AMEs). AMEs change the structure of the aminoglycoside which then prevents their binding to the ribosome. One of the ways AMEs does this is through APHs (nucleoside triphosphate-dependent phosphotransferases). There are over 100 different AMEs described in literature, and 40 crystal structures of 8 different APH enzymes have been analyzed, demonstrating the diversity and variability of this class of enzymes.

APH Structure

The primary, historical method of circumventing AME-mediated resistance has been in the creation of steric hindrance. In this process, the inclusion of precisely placed side chains such as AHB ((S)-4-amino-2-hydroxybutyrate) offer resistance to the actions of many AMEs, a process that has worked effectively in creating new antibiotics like amikacin from kanamycin. Even more recently, Plazomicin has been synthesized by the addition of an AHB group (to position 1 of the 2-deoxystreptamine) and a hydroxyethyl group (to position 6’) to the sisomicin molecule. The effect is an aminoglycoside which appears to be resistant to all but one aminoglycoside resistance enzymes (the exception was AAC(2’)) (Armstrong and Miller, 2010)! There have also been a few others which have shown some efficacy against resistant strains, but in the greater war, that’s not a lot of reserve options. And AMEs are certain to coevolve equally fast with these new antibiotics.

An ideal APH would provide resistance against all members of the AME family. However, that might not be feasible as we learn more about the structures of the different subfamilies of APH enzymes. Researching more specific APH inhibitors might be more useful, but would likely prove less profitable. Considering our inability to produce a lot of next-generation aminoglycosides, however, perhaps specific APH inhibitors are a better choice after all if we want to slow down the impending ARA (Antibiotic Resistance Apocalypse). You can find a more complete review of this subject online from Kun Shi and associates from McGill University.

 
 

Shi, K., Caldwell, S. J., Fong, D. H., & Berghuis, A. M. Prospects for circumventing aminoglycoside kinase mediated antibiotic resistance. Frontiers in Cellular and Infection Microbiology, 3, 22.

Armstrong, E. S., and Miller, G.H. (2010). Combating evolution with intelligent design: the neoglycoside ACHN-490. Curr.
Opin. Microbiol. 13, 565–573.

Category Code: 88221

Posted by Chris on August 22nd, 2013  ⟩  0 comments

Autophagy is a very complicated process when it comes to cancer. In general, autophagy isn’t complicated at all. It is a highly conserved process in which damaged or long-lived proteins and organelles can be removed from cells. Functioning through lysosomal machinery, this process ensures cellular survival during starvation by maintaining cellular energy levels. Think of it as a waste removal/renewable energy source system; bad or unnecessary stuff gets broken down in order to sustain the rest of the cell. This happens in nearly all cells and it works wonderfully.

In cancer, however, the role of autophagy is a bit murkier. Sometimes, autophagy is working with the doctors in the suppression of tumor cells; isolating damaged organelles, allowing cell differentiation or promoting cell death of the cancerous cells. But other times, autophagy is working against the doctors; allowing stressed tumor cells to undergo dormancy and resistance to chemotherapeutic drugs. Such a dichotomy in roles can be very frustrating to the doctors and ultimately deadly to a patient if the therapy is compromised due to the lack of effective autophagy or if the cancer comes back due to effective autophagy. Knowing which cancers use autophagy for survival and which cancers are susceptible to autophagy is key to corrective treatment.

None of this is particularly new, however. The role of autophagy in cancer cells is quite known (there is a dedicated Autophagy Journal, after all). There are also a number of clinical studies on various cancer types which are looking into the use of anti-autophagy drugs in conjunction with chemotherapy drugs. Autophagy-inhibitory drugs can come in one of two types: there are the early-stage inhibitors which can target class III P13K and interfere with its membrane recruitment, and late-stage inhibitors (such as chloroquine, hydrochloroquine and monensin) which prevent the acidification of lysosomes (Yang, 2011). Below is a table of various ongoing (c. 2011) clinical trials utilizing some of these late-stage, autophagy inhibitors.

Autophagy Clinical trials

More recently, a group in South Korea headed by Cheol Hyeon Kim investigated the effect of monensin on two anticancer drugs (erlotinib and rapamycin) in the treatment of lung cancer cells. Erlotinib is an Epidermal Growth Factor Receptor (EGFR) inhibitor which binds to the ATP binding site on the EGFR, which prevents the formation of an EGFR homodimer and its ensuing signaling cascade. Autophagy pathwayRapamycin is a macrocyclic triene, antibacterial drug which also has immunosuppressant and anticancer activities. The major target of rapamycin in mammalian cells is the mTOR pathway, which is a major regulator of autophagy, and which is downstream of the P13K-AKT pathway.

Kim’s group saw an increase in apoptosis in the cells treated with both the anticancer drug as well as nanomolar concentrations of monensin, with a 40% reduction in cells compared to the control and around ~20% reduction compared to the anticancer drug alone (P <0.01). Perhaps this isn’t really that surprisingly, after all. Hydrochloroquine in conjunction with erlotinib is already in a phase 2 trials for lung cancer (see table above). But their published record further assists in the elucidation of how autophagy functions in the realm of cancer cell tumorigenesis and ultimately, that is most important thing right now.

 
 

Choi, H. S., Jeong, E. H., Lee, T. G., Kim, S. Y., Kim, H. R., & Kim, C. H. (2013). Autophagy Inhibition with Monensin Enhances Cell Cycle Arrest and Apoptosis Induced by mTOR or Epidermal Growth Factor Receptor Inhibitors in Lung Cancer Cells. Tuberculosis and Respiratory Diseases, 75(1), 9-17.

Yang, Z. J., Chee, C. E., Huang, S., & Sinicrope, F. A. (2011). The role of autophagy in cancer: therapeutic implications. Molecular cancer therapeutics, 10(9), 1533-1541.

Category Code: 88221 88241