A Few (absolutely hot) Friday Infographics

In the fierce competition for our time and attention, a picture is still worth a thousand words. Infographics are everywhere. Sometimes they can help tell an old story in a new way, sometimes they bring something we never thought about before to our attention, sometimes they are just fun. Here are a few science and non-science infographics I think are worth sharing.

Who knew there was “absolute hot”?

I knew about absolute zero. Until I came across this awesome infographic I had never considered it’s opposite. Now I know.

billion degrees

Continue reading

MicroRNAs as Circulating Biomarkers

12097693_lMicroRNAs (miRNAs) are short strands of RNA averaging between 19-24 nucleotides in length that were first discovered in C.elegans and subsequently shown to exist in species ranging from algae to humans (1). Speculated to be merely “junk” more than a decade ago, miRNAs have emerged as powerful regulators of a wide array of cellular processes because of their influence on gene expression at the posttrancriptional level. Dysregulation of these miRNAs is also associated with life-threatening conditions such as cancer and cardiovascular disease, which points to a potential use of miRNAs in diagnosis and treatment. Recently, it has been demonstrated that miRNAs are present in circulating blood plasma, protected from degradation by inclusion in lipid or lipoprotein complexes. This opens up the possibility to exploit miRNA as a useful diagnostic tool in clinical samples. Continue reading

Reflecting on the Future: Hands-On, Person-to-Person Educational Experiences

iStock_000053412884LargeThe start of a new year is always a good time for reflection.  For those of us at the BioPharmaceutical Technology Center Institute (BTC Institute), this means looking at the programs we offer and considering ones we might like to develop.

In this process, we find ourselves continuing to feel certain that the hands-on, lab-based opportunities we provide add something meaningful to the education of those we serve, from middle school students and their teachers to graduate students to scientists in academia and industry.  The value of learning concepts and techniques in a well-equipped setting, working with teachers and volunteers who are dedicated scientists, is significant.

In addition to gaining an understanding of the basics of molecular biology so key to biotechnology, these programs are also designed to support the development of critical thinking skills so necessary to scientific literacy.

We think this is also the case for our scientific symposia (Wisconsin Stem Cell Symposium; Wisconsin Human Proteomics Symposium) and our International Forum on Consciousness.  These events enable attendees to interact with speakers and other participants in person – in an environment designed to encourage the exchange of information, ideas and perspectives. Continue reading

Mass Spectrometry Application: Antibody Quantitation for Preclinical PK studies

Isoform_Antibodies_LinkedInTherapeutic monoclonal antibodies (mAbs) represent the majority of therapeutics biologics now on the market, with more than 20 mAbs approved as drugs (1–3). During preclinical development of therapeutic antibodies, multiple variants of each antibody are assessed for pharmacokinetic (PK) characteristics across model systems such as rodents, beagles and  primates. Ligand-binding assays (LBA) are the standard technology used to perform the PK studies for mAb candidates (4). Ligand-binding assays (LBAs) are methods used  to detect and measure a macromolecular interaction between a ligand and a binding molecule. In LBAs, a therapeutic monoclonal antibody is considered to be the ligand, or analyte of interest, while the binding molecule is usually a target protein.

LBAs have certain well-documented limitations (5). Specific assay reagents are often not available early in a program. Interferences from endogenous proteins, antidrug antibodies, and soluble target ligands are potential complicating factors.

Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS)-based methods represent a viable and complementary addition to LBA for mAb quantification in biological matrixes. LC–MS/MS provides specificity, sensitivity, and multiplexing capability.

A recent reference (6) illustrates an automated method to perform LC–MS/MS-based quantitation, with IgG1 conserved peptides, a heavy isotope labeled mAb internal standard,and anti-human Fc enrichment. The method was applied to the pharmacokinetic study of a mAb dosed in cynomolgus monkey, and the results were compared with the immunoassay data. The interesting finding of the difference between ELISA and LC–MRM-MS data indicated that those two methods can provide complementary information regarding the drug’s PK profile.

Literature Cited

  1. Mao, T. et al. (2013) Top-Down Structural Analysis of an Intact Monoclonal Antibody by Electron Capture Dissociation-Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry. Anal.Chem. 85, 4239–46.
  2. Weiner, L. M. et al. (2010) Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat. Rev. Immunol. 10, 317–27.
  3. Nelson, A. et al. (2010) Development trends for human monoclonal antibody therapeutics. Nat. Rev. Drug Discovery. 9, 767–74.
  4. DeSilva, B. et al. (2003) Recommendations for the Bioanalytical Method Validation of Ligand-Binding Assays to Support Pharmacokinetic Assessments of MacromoleculesPharm. Res. 20, 1885–00.
  5. Ezan, E.et al. (2009) Critical comparison of MS and immunoassays for the bioanalysis of therapeutic antibodiesBioanalysis 1, 1375–88.
  6. Zhang, Q. et al. (2014) Generic Automated Method for Liquid Chromatography–Multiple Reaction Monitoring Mass Spectrometry Based Monoclonal Antibody Quantitation for Preclinical Pharmacokinetic Studies. Anal.Chem. 86, 8776–84.

What Things Are You Thankful for in Science?

What are you thankful for in science?

What are you thankful for in science?

As the social media lead for Promega, I keep my eye on trends in new media. I have personal accounts that I keep mostly to see what other people are doing. I try hangouts, social networking and other things so that I have an idea of developing practices outside of the biotechnology industry. One activity that has been popular over the last couple of years during the month of November in the United States is the Facebook post of “30 days of thanksgiving”.

I wondered what “thanksgiving” looks like to the research scientist. So I asked:

What are the things you are thankful for in science?

The answers have been as varied as the people I talked to ranging from little things like water bath floats to really big things, like the renewal of your research funding or achieving tenure.

Here are some of the answers from my informal inquiries:

“Tube floaties for water baths.”

—E.V., genomics product manager

“I was always thankful for Geiger counters.”

—K. G., science writer

“Thermal cyclers and Taq Polymerase. As an undergrad I watched someone sit with a timer and move their tubes between water baths at 3 different temperatures, opening tubes and adding polymerase at the end of each cycle. Modern PCR is SOOO much easier.”

—M.M., research scientist

“I am thankful for competent cells. I remember preparing the CaCl2 and doing slow centrifugation. Also thankful for serum-compatible transfection, rapid ligations and online journal access (no longer have to traipse over to the university library to get papers photocopied- uuurrrgggghhh).”

—R.D., technical services scientist

“How about T-vectors for cloning? I was no molecular biologist, but could make a T-vector work.”

—K.K., science writer

“I am thankful for open-access journals and the ability to read the full article without an institutional subscription.”

—S.K., science writer

“I am ever so thankful for ONLINE ORDERING! So awesome. Throw in online technical manuals, on-line support tools, on-line calculators – all are awesome!!”

—A.P., director, scientific courses

“I am thankful for automated sequencing- manual sequencing was laborious and hazardous!!!”

—R.G., technical services scientist

Do any of these resonate with you? What are you thankful for as a scientist? Let us know in the comments.

Tips for Successful Dual-Reporter Assays

Dual-Reporter-AssayRecently, one of my fellow bloggers described some of the advantages of using dual-reporter assays (including our Dual-Luciferase®, Dual-Glo® Luciferase and our new NanoDLR™ assay debuting soon). These assays are relatively easy to understand in principle. Use a primary and secondary reporter vector transiently transfected into your favorite mammalian cell line. The primary reporter is commonly used as a marker for a gene, promoter, or response element of interest. The secondary reporter drives a steady level of expression of a different marker. We can use that second marker to normalize the changes in expression of the primary under the assumption that the secondary marker is unaffected by what is being experimentally manipulated.

While there are many advantages to dual-reporter assays, they require careful planning to avoid common pitfalls. Here’s what you can do to avoid repeating some of the common mistakes we see with new users: Continue reading

Use of Mass Spectrometry to Quantitate Food Allergens

 

Food allergies are becoming increasingly prevalent among children. Credit: James Gathany, CDC

Food allergies are becoming increasingly prevalent among children. Credit: James Gathany, CDC

Food allergies are increasing worldwide and becoming a public health issue, especially among children are concerned. Children have a higher prevalence of food allergies, with about 4–8%, compared to adults (1–5%).  Currently antibody-based methods such ELISA (enzyme-linked immunosorbent assay) are the primary method for food allergen analysis. In most cases antibodies are only available for single well-known allergens. Often those that are commercially available are poorly characterized resulting cross-reactivity that leads to false-positive results in diagnostic tests.

A recent publication (1) presented a review of an alternative technology based on mass spec (i.e., multiple reaction monitoring, MRM) that circumvents the drawbacks of antibody based methods. MRM allows precise quantitative determination of target proteins in complex samples with broad dynamic range.  MRM also provides quantification of different isoforms. It is noted that tryptic digestion followed by mass spec analysis, has already identified several unique peptides for different allergens, including those found in crustaceans, eggs, fish, peanuts, soy and wheat. In summary the challenge is now to select the appropriate tryptic signature peptide(s) for the respective allergen and to develop well characterized standards (i.e., isotope labeled standards) to ensure accurate quanititation.

Citation:

Koeberl, M et al. (2014) Next Generation of Food Allergen Quantification using Mass Spectrometric Systems J. Proteome Research  13, 3499–509.

The Games We Play

Now you can play an RNA folding game (image source RCSB PDB)

Now you can play an RNA folding game (image source RCSB PDB)

Most everyone loves a good puzzle game, and these days there’s an abundance of brain teasers available to us in multiple formats.  Crosswords and Sudoku can still be found in your newspaper, but PC, online, and smartphone puzzles have exploded in recent years.  Recently, I’ve spent many hours trying to level up in Candy Crush Saga (I’m stuck on level 102).  But for me, the best puzzle games are ones that teach science in fun clever ways without making you feel like you’re learning a lesson.

Two of my favorites show you how to design rockets and fly them to planetary bodies: Simple Rockets and Kerbal Space Program (with fun names like “smoon” or “Mun” as known in each respective game).  These games are designed to run on your smartphone, tablet, or PC.  One of my favorites I remember playing in middle school in the late ‘90s, “The Incredible Machine” challenged you to create the most elaborate Rube Goldberg device imaginable.  The new “Contraption Maker” made by the same team will satisfy your urges for elaborate designs for simple problems.

Best of all, puzzle games can do more than just teach what’s already known about science, they can help researchers refine and improve their own research, and generate new science.  Scientists have put our basic desire for new puzzles to use previously in projects like FoldIt where citizen scientists are challenged with folding proteins.

A newer science puzzle game I’m itching to start playing is designed to help scientists understand how RNA folds.  Continue reading

Wobbly Polar Vortex, Kids and Science

ThermometerIf you are living in the northern hemisphere, chances are you have felt the effects of a wobbly polar vortex. Super cold temperatures have plunged southward, bringing with them negative wind chills and closed schools. It’s been too cold to get outside for skiing, building snowmen or sledding. So, those of us with young children are dealing with cabin fever.

Here is a collection of indoor science activities to entertain the most feverish of the cabin bound children. The first set of experiments come from the Kitchen Pantry Scientist. You can find her blog here, with lots of ideas for home brew science. Your children can enjoy learning about the law of conservation of energy by creating Marshmallow Slingshots, or if you want a little art with your science, have some fun with Tie-Dye Milk.

For the younger set, Grover from Sesame Street has a series of five easy experiments that include making pepper run and a glittery volcano. You can even tweet your experimental results using the #5Facts hashtag.

If you want to learn more about the science of all these subzero temperatures, the Kitchen Pantry Scientist has a few experiments for you as well, including a bologna tongue stuck to a flag pole experiment.

So, stuck inside because of the extreme cold? Don’t sweat it. Do some science.