What Will You Do on Your Summer Vacation? Try a BTC Institute Summer Intensive Course

Students and community members enjoy a warm spring day at the Memorial Union Terrace at the University of Wisconsin-Madison on May 8, 2009. ©UW-Madison University Communications 608/262-0067 Photo by: Bryce Richter Date:  05/09    File#:  NIKON D3 digital frame

Students and community members enjoy a warm spring day at the Memorial Union Terrace at the University of Wisconsin-Madison on May 8, 2009.
©UW-Madison University Communications 608/262-0067
Photo by: Bryce Richter
Date: 05/09 File#: NIKON D3 digital frame

Summer, the sweetest season…here in Wisconsin we look forward to warm evenings on the Memorial Union Terrace at UW-Madison, grilling brats and veggie burgers, fresh harvests from the farmer’s market and of course, the BTC Institute’s intensive summer courses.

Here is a brief list of what we are offering this summer –

Neuroscience 675 : Molecular Approaches to Neuroscience (June 15-19; 9am–5:30pm): An introduction to basic molecular biology techniques including cloning, nucleic acid isolation, amplification and analysis; cell-based assays for studying cell death mechanisms and microscopy techniques are presented as well as approaches to studying kinase activity for drug discovery. Guest lectures augment the laboratory-focused, techniques-based curriculum.

Oncology 675 – 001: Core Techniques in Protein and Genetic Engineering (July 13-17;  9am-6pm): An advanced primer on molecular biology techniques; nucleic acid isolation, cloning tools and techniques, PCR technologies (PCR, RT-PCR, qPCR and qRT-PCR), plasmid purification, protein purification techniques, and Western blot form the backbone of this course. Additional topics are included as lectures to provide examples of applications.

 Oncology 675 – 002: Emerging Techniques in Protein and Genetic Engineering (July 20-24; 9am-6pm): Focusing on transcription, translation and especially epigenetics, this course allows students to explore cutting edge techniques used to study molecular biology. Laboratory exercises testing HDAC inhibitor potencies and analyzing cellular consequences of HDAC inhibition, cell health assessment and kinase inhibitor assays for drug profiling, studying protein-protein interactions in live cells, and investigating biologics in live cells. Lectures will tentatively include discussion of deep sequencing and next-gen sequencing, pharmaceutical development, mass spectrometry for discovery, systems biology approaches to experimental design and others.

Enjoy the prairie at the BTC Institute campus and learn something too!

Enjoy the prairie at the BTC Institute campus and learn something too!

Please direct inquiries about the courses to Dr. Amy Prevost (amy.prevost@btci.org) – we hope to see some of you this summer!

Also, visit our web site to see what else we’re doing at the BTC Institute- we have some great summer programs for upper elementary and middle school students, camp biotech for 9th and 10th graders, and  teacher training courses– more information on all of our course offerings at www.btci.org .

10 Bold Summer Activities for Science Lovers

1781140_lAt 25 I often find myself looking for exciting new ideas for enjoying an evening with friends and family that extend beyond the typical dinner, drinks or a movie. Over the three years I have worked at Promega, my interest for scientific discovery has been amplified by the expertise and enthusiasm of my colleagues. In appreciation for science-lovers everywhere, here is a short list of activities for those looking for unique ways of having fun with science this summer.


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Cancer Detection on a Chip?

Figure 5 shows typical scanned images of bead-array for analyzing adjacent normal tissue and tumor tissue. Huang et al. (2015) Digital Detection of Multiple Minority Mutants and Expression Levels of Multiple Colorectal Cancer-Related Genes Using Digital-PCR Coupled with Bead-Array. PLOS ONE 10(4):e0123420. doi:10.1371/journal.pone.0123420.g005

Figure 5 shows typical scanned images of bead-array for analyzing adjacent normal tissue and tumor tissue. Huang et al. (2015) Digital Detection of Multiple Minority Mutants and Expression Levels of Multiple Colorectal Cancer-Related Genes Using Digital-PCR Coupled with Bead-Array. PLOS ONE 10(4):e0123420. doi:10.1371/journal.pone.0123420.g005

The ideal cancer detection method would involve giving a sample of blood or tissue and using DNA or RNA analysis to determine if there were any gene sequence or gene expression changes that are known hallmarks of cancer. Unfortunately, most current screening methods used are not so precise and in some cases are invasive. However useful tests for colon cancer may be, many people do not subject themselves to the standard colonoscopy. What if there was an easier, noninvasive method that could be used to screen for cancer and detect changes at the early, easily treatable stages of cancer? A recent article in PLOS ONE describes just such a mutation detection method for colorectal cancer using purified nucleic acid with a method that involves emulsion PCR, bead arrays and fluorescent probes. Continue reading

Filter-Aided Sample Preparation before Mass Spec Analysis: An Evaluation of FASP and eFASP

12271ma_800pxFilter-aided sample preparation (FASP) method is used for the on-filter digestion of proteins prior to mass-spectrometry-based analyses (1,2). FASP was designed for the removal of detergents, and chaotropes that were used for sample preparation. In addition, FASP removes components such as salts, nucleic acids and lipids. Akylation of reduced cysteine residues is also carried out on filter, after which protein is proteolyzed by use of trypsin on filter in the optimal buffer of the enzyme. Subsequent elution and desalting of the peptide-rich solution then provides a sample ready for LC–MS/MS analysis.

Erde et al. (3) described an enhanced FASP (eFASP) workflow that included 0.2% DCA in the exchange, alkylation, and digestion buffers,thus enhancing trypsin proteolysis, resulting in increases cytosolic and membrane protein representation. DCA has been reported (4) to improve the efficiency of the denaturation, solubilization, and tryptic digestion of proteins, particularly proteolytically resistant myoglobin and integral membrane proteins, thereby enhancing the efficiency of their identification with regard to the number of identified proteins and unique peptides.

In a recent publication (5) traditional FASP and eFASP were re-evaluated by ultra-high-performance liquid chromatography coupled to a quadrupole mass filter Orbitrap analyzer (Q Exactive). The results indicate that at the protein level, both methods extracted essentially the same number of hydrophobic transmembrane containing proteins as well as proteins associated with the cytoplasm or the cytoplasmic and outer membranes.

The LC–MS/MS results indicate that FASP and eFASP showed no significant differences at the protein level. However, because of the slight differences in selectivity at the physicochemical level of peptides, these methods can be seen to be somewhat complementary for analyses of complex peptide mixtures.

  1. Manza, L. L. et al. (2005) Sample preparation and digestion for proteomic analyses using spin filters Proteomics  5, 1742–74.
  2. Wiśniewski, J. R. et al. (2009) Universal sample preparation method for proteome analysis Nat. Methods 6, 359–62.
  3. Erde, J. et al. (2014) Enhanced FASP (eFASP) to increase proteomic coverage and sample recovery for quantitative proteome experiments. J. Proteome Res. 13, 1885–95.
  4. Lin, Y. et al. (2008) Sodium-deoxycholate-assisted tryptic digestion and identification of proteolytically resistant proteins Anal. Biochem.  377, 259–66.
  5. Nel. A. et al. (2015) Comparative Reevaluation of FASP and Enhanced FASP methods by LC-MS/MS/ J Proteome Res. 14, 1637–42.

Your Health has a Season

Photo of pasque flowers

Pasque flowers in a northern hemisphere garden in spring.

As the seasons change so does the general state of health for many of us. The further from the equator we live, the more pronounced these effects are. For instance, did you know that blood pressure elevation for many people increases with the distance they live from the equator, an effect most pronounced during the low sunlight season (winter in the northern hemisphere)?

A report published online in Nature Communications May 12, shows evidence of changes in cellular physiology with the seasons. Todd et al. published a study entitled: “Widespread seasonal gene expression reveals annual differences in human immunity and physiology”, where they note,

“Here we find more than 4,000 protein-coding mRNAs in white blood cells and adipose tissue to have seasonal expression profiles, with inverted patterns observed between Europe and Oceania.”

Let’s Take a Look at the Research

Todd et al. looked at ethnically and geographically distinct populations, including subjects from Australia, The Gambia (Africa), Germany, the UK and Iceland. Individuals from the various studies were infants, adults with type 1 diabetes and asthmatics in the range of 18-83 years of age. The authors analyzed RNA from peripheral blood mononuclear cells and subcutaneous adipose tissue biopsies, as well as examining peripheral blood cell counts and circulating levels of proinflammatory cytokines. Continue reading

Targeting MYC: The Need to Study Protein:Protein Interactions in Cells

Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;

Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;

In 1982, picked up because of its homology to chicken virus genes that could transform cells, MYC became one of the first human genes identified that could drive cellular transformation (1,2). Since that time countless laboratories have prodded and poked the human MYC gene, the MYC protein, their homologs in other animal models, and their transforming viral counterparts.

MYC is a transcription factor and forms heterodimers with a required protein partner, MAX, before binding to the E box sequences of DNA regulatory regions (3). MYC regulates gene expression of many targets through interactions with a host of proteins, often referred to as the MYC Interactome (2).  In fact, MYC is estimated to bind 10–15% of the genome, and it regulates the expression of genes that  are transcribed by by each of the three RNA polymerases (2).

MYC plays a central role in regulating cell growth, proliferation, apoptosis, differentiation and transformation, acting as a central integrator of cellular signals. MYC is tightly regulated at multiple levels from gene expression to protein stability. Dysregulation (usually upregulation) of the amount and stability of Myc protein is observed in many human cancers. Even in cancers in which MYC is not directly involved in transforming cells, its normal expression is often required to support the extracellular matrix and/or vascularization necessary for tumor growth and formation (4).

Because MYC is such a central player cancer pathology, it is an attractive target for cancer therapeutics  (2) . Continue reading

Compelling Science Communication

An archive of 35mm slides. There's probably one in a dark corner of your lab.

An archive of 35mm slides. There’s probably one in a dark corner of your lab.

Back in the dark ages, when I was in graduate school, if we were traveling to a conference to give a presentation, we always made sure that our slide carousels (yes, scientific talks used to be given from 35 mm slides) were in our carry-on baggage. That carousel was more important than our underwear or our toothbrush, no trusting it to baggage claim.

Things have improved markedly, I’m happy to say. Now everything has to go in the carry-on baggage, because most PIs don’t have the financial room in their grants to pay for checked bag fees. Fortunately, we can store copies of our presentations on several different clouds and bring a thumb drive or two on board the plane, tucked safely away in the underwear in the duffle bag that doesn’t quite fit in the overhead compartment. No need to choose between unwieldy slide carousels and clothes.

But are PowerPoint® and Prezi® presentations the best way to communicate your science? When you hit your audience with slide after slide of bullets are you killing their interest? When you show that slide of three years worth of work and say “Don’t worry about trying to read this…” are you killing your presentation?

Is there a better more compelling way to communicate science? Accurately. So that people care. So that people understand.

FameLab International certainly thinks so. Begun in 2005 by the Cheltenham Science Festival in the UK, FameLab seeks to promote better science communication through sponsorship of a competition in which scientists and engineers have three short minutes to communicate their science with enthusiasm and accuracy–armed “with only their wits and a few props that they can carry on stage.”

It is truly a global competition with over 5,000 young scientists and engineers from 25 countries around the world competing for the grand prize each year. The Grand Final Competition is held in June each year, but you can take a sneak peak at some of the entrants on the FameLab Facebook page now.

Here’s a winning taste from the 2014 competition. There is more on the FameLab YouTube channel.

From Death Row to Exoneration Thanks to DNA Testing

Hands of man prisoner gripping rusty prison barsImagine being convicted of a crime for which you are not guilty—not some minor crime, but one of the most heinous crimes imaginable: the rape and murder of a young girl. Would you feel shock and anger at the injustice? Disappointment in the legal system that could make such a horrible error? Sadness and depression at the thought of spending time imprisoned for a crime that someone else committed? Probably all of those emotions and more. At your sentencing hearing, the situation gets worse; you are sentenced to death. Now, this horrible crime will prematurely claim the life of two innocents: the young girl and you.

This is the situation that Kirk Bloodsworth faced in 1985: a death sentence for the rape and murder of 9-year-old Dawn Hamilton. Although Bloodsworth didn’t know it at the time, DNA testing would eventually prove his innocence and save his life.
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Take Notes and Graduate Faster!

Cell density illustrationOne piece of advice you will get from our Technical Services and R&D Scientists with regard to cell-based assays is to pay attention to what you are doing. Sounds obvious, but sloppiness can easily enter into the equation. Do you always count your viable cells with a hemocytometer and trypan blue exclusion before you split a culture? Do you always make sure that each well of your plate or plates contain the same number of cells? Two of our scientists, Terry Riss and Rich Moravec, published a paper demonstrating how decisions you make in experimental setup can ultimately affect the results you obtain. A natural consequence of this is difficulty replicating experiments if you didn’t pay attention to the details during the initial experimental setup.

Cell Density Per Well Affects Response to Treatment
To demonstrate how cell density can affect your data, Riss and Moravec set up parallel plates with three different cell densities of HepG2 cells and measured the response to tamoxifen. The lower the cell density per well, the more pronounced the effect of the tamoxifin on the cells. Higher density cells were more resistant to tamoxifen. Continue reading

Choosing Primary and Control Reporters for Dual-Luciferase Assays

Dual-Reporter Assays give scientists the ability to simultaneously measure two reporter enzymes within a single sample. In dual assays, the activity of an experimental reporter is correlated with the effect of specific experimental conditions, while the activity of a control reporter relays the baseline response, providing an essential internal control that reduces variability caused by differences in cell viability or transfection efficiency. The Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay provides a choice of two sensitive reporters (firefly and NanoLuc luciferases) for use in dual-assay format. Both reporters give state-of-the-art functionality, raising the question “Which luciferase should be the primary reporter and which should be the control?”

This infographic outlines the various NanoDLR dual-reporter assay choices and the situations where you would choose one format over another. Continue reading