“Reverse” Molecular Reactions in DNA through Mind-Body Interventions

While my morning routine typically only involves a large cup of coffee, increasingly more Americans are beginning their days with a set of sun salutations. Sun salutations are a series of poses originating from yoga, one of the most popular types of mind-body intervention in the United States. Along with yoga, other commonly recognized mind-body interventions (MBI) include meditation, mindfulness, Tai chi, and Qigong. Despite the fact that each of these activities differ in the amount of physical effort required, they all view mental and physical health as single cohesive system.

The influence of overall mind-body intervention on health and wellness is an ancient concept that is now revolutionizing Western medicine. In the past, Western medicine has focused primarily on the health of the physical body. Yoga and meditation were viewed as beneficial, but were less likely to be recommended by clinicians as a method for relief. Now, with recent developments in gene expression analysis techniques, we have a better understanding of biological mechanisms and how they interact with psychological variables. A possible shift in clinician’s philosophies can be seen in the steady rise in the complementary health approaches of yoga, Tai chi, and qi gong1.

To completely understand how MBI affects a person’s health, we must first realize the links between stress and the conserved transcriptional response to adversity (CTRA). CTRA refers to the common molecular pattern discovered in individuals facing hardship. Whether it be in the form of diagnosis of a life-threatening disease or the death of a loved one, the characteristics of CTRA stay consistent. CTRA causes an influx in the production of epinephrine and norepinephrine. These neuromodulators then affect the production of transcription factors. Continue reading

Welcome to Your Biotechnology Field Trip at the BTC Institute!

Kaukauna High School students arrive at the BTC for a biotechnology fieldtrip.

Kaukauna High School students arrive at the BTC for a biotechnology fieldtrip.

BTCI provides our students an opportunity that they could never get in the classroom.
—Jim Geoffrey, Biology Teacher, Kaukauna High School

Your bus has arrived and parked in the circular driveway at the front of the BioPharmaceutical Technology Center on the Promega Corporation campus in Fitchburg, WI. Your BTC Institute hosts – and instructors – for your field trip are Barbara Bielec (K-12 Program Director) and Ryan Olson (Biotechnology Instructor). They’ll greet you in the Atrium and direct you to a conference room where you can leave coats and backpacks, and then to the lab you’ll be working in during your visit.

Here’s a taste of what happened next for students from Random Lake High School and Wonewoc High School on December 3rd, and from Kaukauna High School on December 4th. Continue reading

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.